Method to identify subjects at higher risk to develop an autoimmune disease based on genetic and/or phenotypic screening for epistatic variants in ddx39b (rs2523506) and il7r (rs6897932)

ABSTRACT

The present invention includes a method, kits, and assays for identifying a human subject as having an increased risk of developing an autoimmune disease, or a human subject with multiple sclerosis caused by elevated soluble Interleukin 7 receptor (sIL7R), by obtaining a biological sample and detecting or measuring in the biological sample an amount of a soluble Interleukin-7 receptor (sIL7R) and an amount of an RNA Helicase DDX39B, whereby a lower expression of DDX39B and a higher secretion of sIL7R identifies the subject from which the biological sample was obtained as having an increased risk of developing an autoimmune disease, when compared to a human subject not having an autoimmune disease. The present invention also includes a method of modifying a treating of subjects based on the lower expression of RNA Helicase DDX39B alone or in combination with an increase in sIL7R.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Ser. No. 15/928,939, filed Mar.22, 2018 and claims benefit to U.S. Provisional Application Ser. No.62/474,951, filed Mar. 22, 2017, the entire contents of which areincorporated herein by reference.

STATEMENT OF FEDERALLY FUNDED RESEARCH

This invention was made with government support under R01-NS060925, andF32-NS087899, awarded by NIH/NINDS. The government has certain rights inthe invention.

TECHNICAL FIELD OF THE INVENTION

The present invention relates in general to the field of genetic orphenotypic testing for risk of autoimmune disorders, and moreparticularly, to a novel method for identifying subjects at risk formultiple sclerosis.

BACKGROUND OF THE INVENTION

Without limiting the scope of the invention, its background is describedin connection with multiple sclerosis.

Multiple Sclerosis (MS) is a chronic autoimmune disorder characterizedby self-reactive T cell-mediated damage to neuronal myelin sheaths inthe central nervous system (CNS) that leads to axonal demyelination,neuronal death and progressive neurological dysfunction. Up to date,there is no cure for the disease and available treatments can only slowdown disease progression, often by suppressing the immune system.Unfortunately, patients are normally diagnosed after manifestation ofclinical symptoms, at which time the patient has suffered substantialneuronal damage and this cannot be reverted with current approvedtreatments. Therefore, there is an unmet need for early detection anddiagnosis of MS.

The breach of immunological tolerance that leads to MS is thought tooriginate from complex interactions between environmental and geneticfactors. Under this view, the genetic background of an individual couldgenerate an environment permissive for the survival of self-reactivelymphocytes, which could be subsequently activated by the presence of anenvironmental trigger, usually in the form of viral or bacterialinfection. Accordingly, methods for genetic screening of variantsassociated with increased MS risk could provide a valuable tool toidentify individuals at higher risk to develop MS.

U.S. Pat. No. 8,158,344 shows that a driver of increased MS risk is thesoluble form of the interleukin-7 receptor alpha chain gene (sIL7R),produced by alternative splicing of IL7R exon 6. The present inventorsand others have previously shown that the variant rs6897932 (C/T, whereC is the risk allele) within IL7R exon 6 is strongly associated withincreased MS risk (Gregory et al., 2007; International MultipleSclerosis Genetics et al., 2007; Lundmark et al., 2007). Furthermore,the present inventors showed that the risk ‘C’ allele of this variantincreases skipping of the exon (Evsyukova et al., 2013; Gregory et al.,2007), leading to up-regulation of sIL7R (Hoe et al., 2010; Lundstrom etal., 2013). Importantly, sIL7R has been shown to exacerbate the clinicalprogression and severity of the disease in the Experimental AutoimmuneEncephalomyelitis (EAE) mouse model of MS, presumably by potentiatingthe bioavailability and/or bioactivity of IL-7 cytokine (Lundstrom etal., 2013). Collectively, these data link alternative splicing of IL7Rto the pathogenesis of MS, and U.S. Pat. No. 8,158,344 describes methodsfor screening based on the effects of IL7R rs6897932.

SUMMARY OF THE INVENTION

In one embodiment, the present invention includes a method ofidentifying a human subject as having an increased risk of developing anautoimmune disease caused by lower levels of an RNA Helicase DDX39B,comprising: obtaining a biological sample from a subject suspected ofhaving an autoimmune disease; and detecting or measuring in thebiological sample an amount of an RNA Helicase DDX39B, whereby a lowerexpression of DDX39B identifies the subject from which the biologicalsample was obtained as having an autoimmune disease or having increasedrisk of developing an autoimmune disease, when compared to a humansubject not having an autoimmune disease. In one aspect, the methodfurther comprises the step of measuring the expression levels of thesoluble isoform of the Interleukin-7 Receptor (sIL7R), wherein a highersecretion of sIL7R and a lower expression of DDX39B identifies thesubject from which the biological sample was obtained as having anautoimmune disease or having increased risk of developing an autoimmunedisease, when compared to a human subject not having an autoimmunedisease because DDX39B is a critical splicing factor required forinclusion of exon 6 in IL7R pre-mRNAs. In another aspect, the autoimmunedisease is selected from Multiple sclerosis, Type I diabetes, Rheumatoidarthritis, Systemic lupus erythematosus, Atopic dermatitis, Ankylosingspondylitis, Primary biliary cirrhosis, or inflammatory bowel syndromessuch as Ulcerative colitis and Crohn's disease. In another aspect, thestep of detecting or measuring in the biological sample is definedfurther as being selected from at least one of: detecting a presence ofthe risk alleles at SNPs associated with multiple sclerosis in DDX39Band IL7R genes, at least one SNP selected from at least rs6897932 andrs2523506, or any allele in linkage disequilibrium with the DDX39B andIL7R MS risk alleles; a differential expression of IL7R RNA isoforms; adifferential expression of IL7R protein isoforms; a differentialexpression of DDX39B protein; or any combination thereof; detecting ormeasuring in the biological sample is defined further as detectingallelic variants in DDX39B nucleic acids, which encodes an RNA helicasecritical for inclusion of exon 6 in the Interleukin-7 receptor (IL7R)mRNA, whereby the presence of the risk A allele at the SNP rs2523506 inDDX39B exon 1 (5′ UTR of DDX39B mRNAs), or the presence of thecomplementary allele in the opposite strand, or the presence of anyother allele in linkage disequilibrium with rs2523506, identifies thesubject from which the biological sample was obtained as having multiplesclerosis or having an increased risk of developing multiple sclerosis,relative to a biological sample from a human subject lacking the riskallele at the SNP rs2523506; detecting or measuring in the biologicalsample is defined further as detecting allelic variants in DDX39B andthe Interleukin-7 receptor (IL7R), whereby the presence of the risk Aallele at the SNP rs2523506 in DDX39B exon 1 (5′ UTR of DDX39B mRNAs)and the presence of the risk C allele at the SNP rs6897932 in IL7R exon6, or the presence of the complementary allele in the opposite strand,or the presence of any other allele in linkage disequilibrium with atleast one of rs2523506 or rs6897932, identifies the subject from whichthe biological sample was obtained as having multiple sclerosis or anincreased risk of developing multiple sclerosis, relative to abiological sample from a human subject lacking the risk allele at theSNPs rs2523506 and/or rs6897932; detecting or measuring in thebiological sample is defined further as detecting phenotypic differencesin the expression of Interleukin-7 receptor (IL7R) mRNA isoforms and RNAHelicase DDX39B, whereby an elevated fraction of IL7R mRNAs that lackexon 6 in the biological sample from an individual carrier of the riskalleles at rs6897932 and/or rs2523506, or any other variant in linkagedisequilibrium with rs6897932 and/or rs2523506, identifies the subjectfrom which the biological sample was obtained as having multiplesclerosis or an increased risk of developing multiple sclerosis,relative to a biological sample from a human subject lacking the riskallele at the SNPs rs2523506 and/or rs6897932; detecting or measuring inthe biological sample is defined further as detecting phenotypicdifferences in the expression of Interleukin-7 receptor (IL7R) proteinisoforms and RNA Helicase DDX39B, whereby elevated levels of the solubleform of IL7R (sIL7R) in the biological sample from an individual carrierof the risk alleles at either rs6897932 and/or rs2523506, or any othervariant in linkage disequilibrium with rs6897932 or rs2523506,identifies the subject from which the biological sample was obtained ashaving multiple sclerosis or an increased risk of developing multiplesclerosis, relative to a biological sample from a human subject lackingthe risk allele at the SNPs rs2523506 and/or rs6897932; detecting ormeasuring in the biological sample is defined further as detectingphenotypic differences in the expression of DDX39B protein in a subjectsuspected of having multiple sclerosis, whereby decreased expression ofDDX39B protein in the biological sample identifies the subject fromwhich the biological sample was obtained as having multiple sclerosis oran increased risk of developing multiple sclerosis, relative to abiological sample from a subject not suspected to have multiplesclerosis; or detecting or measuring in the biological sample is definedfurther as detecting phenotypic differences in the expression of DDX39Bprotein, whereby decreased expression of DDX39B protein in thebiological sample from an individual carrier of the risk allele atrs2523506, or any other variant in linkage disequilibrium withrs2523506, identifies the subject from which the biological sample wasobtained as having multiple sclerosis or an increased risk of developingmultiple sclerosis, relative to a biological sample from a human subjectlacking the risk allele at the SNPs rs2523506. In another aspect, thestep of detecting or measuring in the biological sample is a detectionof nucleic acids by a hybridization reaction, a polymerase chainreaction, restriction endonuclease digestion analysis, restrictionfragment length polymorphism (RFLP) analysis, an amplification reaction,an isothermal amplification reaction, or a multiplex amplificationreaction, a polymerase chain reaction (PCR) amplification reaction, areal-time quantitative polymerase chain reaction (qPCR) amplificationreaction, a reverse transcriptase PCR (RT-PCR) amplification reaction,primer extension, DNA array technology, a linear amplificationtechnique, a ligation reaction, direct sequencing, a sequencingreaction, or a combination thereof. In another aspect, the step ofdetecting or measuring in the biological sample is a detection of IL7Ror RNA Helicase DDX39B proteins by LUMINEX, ELISA, immunoassay, massspectrometry, high performance liquid chromatography, two-dimensionalelectrophoresis, Western blotting, flow cytometry, chemiluminescenceimmunoassay, a sandwich assay, a precipitation reaction, animmunoprecipitation reaction, a precipitin reaction, a gel diffusion,immunodiffusion assay, an agglutination assay, a fluorescentimmunoassay, protein microarray, radioimmunoassay, or antibodymicroarray. In another aspect, the method further comprises detecting anmRNA for Interleukin-7 receptor (IL7R) exon 6 splice variants in thebiological sample. In another aspect, the method further comprisesdifferentiating between a subject having an increased risk of multiplesclerosis or as having multiple sclerosis. In another aspect, the methodfurther comprises detecting DDX39B interaction with ESE2 that promotesinclusion of IL7R exon 6, and decreases sIL7R expression, which isindicative of a reduced risk for multiple sclerosis. In another aspect,the method further comprises detecting the presence of the risk alleleat rs2523506 in the 5′ UTR of DDX39B, which reduces translation ofDDX39B mRNAs and increases MS risk.

In one embodiment, the present invention includes a method ofidentifying a human subject as having an increased risk of developing anautoimmune disease caused by elevated levels of soluble Interleukin-7Receptor (sIL7R) and lower levels of an RNA Helicase DDX39B, comprising:obtaining a biological sample from a subject suspected of having anautoimmune disease; and detecting or measuring in the biological samplean amount of a soluble Interleukin-7 receptor (sIL7R) and an amount ofan RNA Helicase DDX39B, whereby a higher secretion of sIL7R and a lowerexpression of DDX39B identifies the subject from which the biologicalsample was obtained as having an autoimmune disease or having increasedrisk of developing an autoimmune disease, when compared to a humansubject not having an autoimmune disease.

In yet another embodiment, the present invention includes an assaycomprising: measuring an interaction between DDX39B rs2523506 and IL7Rrs6897932 by: obtaining a biological sample; detecting in the biologicalsample an amount of a soluble Interleukin-7 receptor (sIL7R) and anamount of an RNA Helicase DDX39B. In one aspect, a higher secretion ofsIL7R and/or lower expression of DDX39B identifies the subject fromwhich the biological sample was obtained as having an increased risk ofdeveloping an autoimmune disease, when compared to a human subject nothaving an autoimmune disease. In another aspect, the autoimmune diseaseis selected from Multiple sclerosis, Type I diabetes, Rheumatoidarthritis, Systemic lupus erythematosus, Atopic dermatitis, Ankylosingspondylitis, Primary biliary cirrhosis, or inflammatory bowel syndromessuch as Ulcerative colitis and Crohn's disease. In another aspect, thelevels of the sIL7R and RNA Helicase DDX39B are compared to a subjectthat does not have an autoimmune disease, wherein an increase in sIL7Ror a decrease in RNA Helicase DDX39B are indicative of an increase riskof the subject having an autoimmune disease. In another aspect, the stepof detecting or measuring in the biological sample is defined further asbeing selected from: detecting a presence of the risk alleles at SNPsassociated with multiple sclerosis in DDX39B and IL7R genes, at leastone SNP selected from at least rs6897932 and rs2523506, or any allele inlinkage disequilibrium with the DDX39B and IL7R MS risk alleles; adifferential expression of IL7R RNA isoforms; a differential expressionof IL7R protein isoforms; a differential expression of DDX39B protein;or any combination thereof; detecting or measuring in the biologicalsample is defined further as detecting allelic variants in DDX39Bnucleic acids, which encodes an RNA helicase critical for inclusion ofexon 6 in the Interleukin-7 receptor (IL7R) mRNA, whereby the presenceof the risk A allele at the SNP rs2523506 in DDX39B exon 1 (5′ UTR ofDDX39B mRNAs), or the presence of the complementary allele in theopposite strand, or the presence of any other allele in linkagedisequilibrium with rs2523506, identifies the subject from which thebiological sample was obtained as having multiple sclerosis or having anincreased risk of developing multiple sclerosis, relative to abiological sample from a human subject lacking the risk allele at theSNP rs2523506; detecting or measuring in the biological sample isdefined further as detecting allelic variants in DDX39B and theInterleukin-7 receptor (IL7R), whereby the presence of the risk A alleleat the SNP rs2523506 in DDX39B exon 1 (5′ UTR of DDX39B mRNAs) and thepresence of the risk C allele at the SNP rs6897932 in IL7R exon 6, orthe presence of the complementary allele in the opposite strand, or thepresence of any other allele in linkage disequilibrium with at least oneof rs2523506 or rs6897932, identifies the subject from which thebiological sample was obtained as having multiple sclerosis or anincreased risk of developing multiple sclerosis, relative to abiological sample from a human subject lacking the risk allele at theSNPs rs2523506 and/or rs6897932; detecting or measuring in thebiological sample is defined further as detecting phenotypic differencesin the expression of Interleukin-7 receptor (IL7R) mRNA isoforms,whereby an elevated fraction of IL7R mRNAs that lack exon 6 in thebiological sample from an individual carrier of the risk alleles atrs6897932 and rs2523506, or the presence of the complementary allele inthe opposite strand, or any other variant in linkage disequilibrium withrs6897932 and/or rs2523506, identifies the subject from which thebiological sample was obtained as having multiple sclerosis or anincreased risk of developing multiple sclerosis, relative to abiological sample from a human subject lacking the risk allele at theSNPs rs2523506 and/or rs6897932; detecting or measuring in thebiological sample is defined further as detecting phenotypic differencesin the expression of Interleukin-7 receptor (IL7R) protein isoforms,whereby elevated levels of the soluble form of IL7R (sIL7R) in thebiological sample from an individual carrier of the risk allelesrs6897932 and rs2523506, or the presence of the complementary allele inthe opposite strand, or any other variant in linkage disequilibrium withrs6897932 or rs2523506, identifies the subject from which the biologicalsample was obtained as having multiple sclerosis or an increased risk ofdeveloping multiple sclerosis, relative to a biological sample from ahuman subject lacking the risk allele at the SNPs rs2523506 and/orrs6897932; detecting or measuring in the biological sample is definedfurther as detecting phenotypic differences in the expression of DDX39Bprotein in a subject suspected of having multiple sclerosis, wherebydecreased expression of DDX39B protein in the biological sampleidentifies the subject from which the biological sample was obtained ashaving multiple sclerosis or an increased risk of developing multiplesclerosis, relative to a biological sample from a subject not suspectedto have multiple sclerosis; or detecting or measuring in the biologicalsample is defined further as detecting phenotypic differences in theexpression of DDX39B protein, whereby decreased expression of DDX39Bprotein in the biological sample from an individual carrier of the riskallele at rs2523506, or any other variant in linkage disequilibrium withrs2523506, identifies the subject from which the biological sample wasobtained as having multiple sclerosis or an increased risk of developingmultiple sclerosis, relative to a biological sample from a human subjectlacking the risk allele at the SNPs rs2523506. In one aspect, the assaydetects or measures in the biological sample is a detection of nucleicacids by a hybridization reaction, a polymerase chain reaction,restriction endonuclease digestion analysis, restriction fragment lengthpolymorphism (RFLP) analysis, an amplification reaction, an isothermalamplification reaction, or a multiplex amplification reaction, apolymerase chain reaction (PCR) amplification reaction, a real-timequantitative polymerase chain reaction (qPCR) amplification reaction, areverse transcriptase PCR (RT-PCR) amplification reaction, primerextension, DNA array technology, a linear amplification technique, aligation reaction, direct sequencing, a sequencing reaction, or acombination thereof. In one aspect, the assay detects or measures in thebiological sample is a detection of IL7R or proteins RNA Helicase DDX39Bby LUMINEX, ELISA, immunoassay, mass spectrometry, high performanceliquid chromatography, two-dimensional electrophoresis, Westernblotting, flow cytometry, chemiluminescence immunoassay, a sandwichassay, a precipitin reaction, an immunoprecipitation reaction, a geldiffusion immunodiffusion assay, an agglutination assay, a fluorescentimmunoassay, protein microarray, radioimmunoassay, or antibodymicroarray. In another aspect, the assay also comprises a display thatshows differentiating between a subject having an increased risk ofmultiple sclerosis or as having multiple sclerosis. In another aspect,an allelic variant of the DDX39B gene is rs2523506 or any other variantin linkage disequilibrium. In another aspect, the assay detects DDX39Bprotein binding to ESE2 that promotes inclusion of IL7R exon 6, anddecreases sIL7R expression, which is indicative of a reduced risk formultiple sclerosis. In another aspect, the assay detects the presence ofthe risk allele at rs2523506 in the 5′ UTR of DDX39B, which reducestranslation of DDX39B mRNAs and increases MS risk.

Yet another embodiment of the present invention includes a kit formeasuring an RNA Helicase DDX39B, comprising: a container comprising afirst agent for the detection of an amount of an RNA Helicase DDX39B;and instructions for determining the amount of the first agent. In oneaspect, the kit further comprises instructions for determining whetherthe amount of at least the first agent in a biological sample from asubject that has or is suspected of having an autoimmune disease isgreater or lower than an amount in a biological sample from a subjectthat does not have or is not suspected of having an autoimmune disease.In another aspect, the kit further comprises reagents for detection ofnucleic acids of the RNA Helicase DDX39B by a hybridization reaction, apolymerase chain reaction, restriction endonuclease digestion analysis,restriction fragment length polymorphism (RFLP) analysis, anamplification reaction, an isothermal amplification reaction, or amultiplex amplification reaction, a polymerase chain reaction (PCR)amplification reaction, a real-time quantitative polymerase chainreaction (qPCR) amplification reaction, a reverse transcriptase PCR(RT-PCR) amplification reaction, primer extension, DNA array technology,a linear amplification technique, a ligation reaction, directsequencing, a sequencing reaction, or a combination thereof. In anotheraspect, the kit further comprises reagents for detection of the RNAHelicase DDX39B in the biological sample by LUMINEX, ELISA, immunoassay,mass spectrometry, high performance liquid chromatography,two-dimensional electrophoresis, Western blotting, flow cytometry,chemiluminescence immunoassay, a sandwich assay, a precipitationreaction, an immunoprecipitation reaction, precipitin reaction, a geldiffusion immunodiffusion assay, an agglutination assay, a fluorescentimmunoassay, protein microarray, radioimmunoassay, or antibodymicroarray. In another aspect, the kit further comprises reagents fordetection of a second agent, wherein the second agent is a pre-mRNA,RNA, or protein of the soluble IL7R or the membrane IL7R, wherein thedetection is at the nucleic acid or protein level.

Yet another embodiment of the present invention includes a method ofidentifying the activity of an RNA Helicase DDX39B, comprising:obtaining a biological sample; and detecting or measuring in thebiological sample an amount of an RNA Helicase DDX39B, its binding, orits activity. In one aspect, the method further comprises measuring anexpression of a soluble Interleukin-7 receptor (sIL7R), wherein thecombination of a higher secretion of sIL7R and a decrease in theexpression or activity of RNA Helicase DDX39B is determined. In anotheraspect, the amount of an RNA Helicase DDX39B is measured by detecting aratio of IL7R mRNA isoforms including or excluding exon 6, or a ratio ofthe resulting IL7R protein isoforms, or a detectable agent under controlof the sequences that control splicing of IL7R exon 6. In anotheraspect, the step of detecting or measuring in the biological sample isdetection of nucleic acids of the Interleukin-7 receptor mRNA lackingexon 6 and the RNA Helicase DDX39B by a hybridization reaction, apolymerase chain reaction, restriction endonuclease digestion analysis,restriction fragment length polymorphism (RFLP) analysis, anamplification reaction, an isothermal amplification reaction, or amultiplex amplification reaction, a polymerase chain reaction (PCR)amplification reaction, a real-time quantitative polymerase chainreaction (qPCR) amplification reaction, a reverse transcriptase PCR(RT-PCR) amplification reaction, primer extension, DNA array technology,a linear amplification technique, a ligation reaction, directsequencing, a sequencing reaction, or a combination thereof. In anotheraspect, the step of detecting or measuring in the biological sample is adetection of IL7R protein isoforms and RNA Helicase DDX39B protein byLUMINEX, ELISA, immunoassay, mass spectrometry, high performance liquidchromatography, two-dimensional electrophoresis, Western blotting,chemiluminescence immunoassay, a sandwich assay, a precipitationreaction, an immunoprecipitation reaction, precipitin reaction, a geldiffusion immunodiffusion assay, an agglutination assay, a fluorescentimmunoassay, protein microarray, radioimmunoassay, or antibodymicroarray.

Yet another embodiment of the present invention includes a method oftreating a subject with an autoimmune disease caused by lower levels ofan RNA Helicase DDX39B, comprising: obtaining a biological sample from asubject suspected of having an autoimmune disease; detecting ormeasuring in the biological sample an amount of an RNA Helicase DDX39B,wherein a patient is elected for treatment if they have a lowerexpression or activity of the RNA Helicase DDX39B; and selecting atreatment for the subject from any of the currently availabletreatments, such as, e.g., mitoxatrone, interferon beta-1a,PEG-interferon beta-1a, azathioprine, fingolimod, natalizumab, ormethylprednisone.

Yet another embodiment of the present invention includes a method oftreating a subject with an autoimmune disease caused by elevated levelsof the soluble isoform of interleukin-7 receptor (sIL7R), comprising:obtaining a biological sample from a subject suspected of having anautoimmune disease; detecting or measuring in the biological sample anamount of the soluble interleukin-7 receptor (sIL7R) and Multiplesclerosis risk alleles in DDX39B rs2523506 and IL7R rs6897932, or thecomplementary allele in the opposite strand, or any other allele inlinkage disequilibrium with rs2523506 and/or rs6897932, wherein apatient is elected for treatment if they have a elevated expression ofthe soluble interleukin-7 receptor (sIL7R) and risk alleles in DDX39Band/or IL7R; and selecting a treatment for the subject from any of thecurrently available treatments such as, e.g., mitoxatrone, interferonbeta-1a, PEG-interferon beta-1a, azathioprine, fingolimod, natalizumab,or methylprednisone.

Yet another embodiment of the present invention includes a method oftreating a subject with an autoimmune disease caused by elevated levelsof the soluble isoform of interleukin-7 receptor (sIL7R), comprising:obtaining a biological sample from a subject suspected of having anautoimmune disease; detecting or measuring in the biological sample anamount of the soluble interleukin-7 receptor (sIL7R) and an amount of anRNA Helicase DDX39B, wherein a patient is elected for treatment if theyhave a elevated expression of the soluble interleukin-7 receptor (sIL7R)and lower expression or activity of the RNA Helicase DDX39B; andselecting a treatment for the subject from any of the currentlyavailable treatments such as, e.g., mitoxatrone, interferon beta-1a,PEG-interferon beta-1a, azathioprine, fingolimod, natalizumab, ormethylprednisone.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures and in which:

FIGS. 1A to 1F show that DDX39B regulates alternative splicing of IL7Rexon 6. FIGS. 1A-1D shows Knockdown of DDX39B in HeLa cells using twoindependent DDX39B siRNAs (DDX_3 and DDX_4) and a non-silencing controlsiRNA (NSC). FIG. 1A, Western blot analysis illustrating depletion ofDDX39B. FIGS. 1B-1C, RT-PCR analysis of IL7R exon 6 splicing (+E6=exonincluded; −E6=exon skipped) in transcripts from a reporter minigene(FIG. 1B) or the endogenous gene (FIG. 1C). FIG. 1D shows thequantification of sIL7R secretion by ELISA. FIGS. 1E-1F shows rescueexperiments with HeLa cell lines stably expressing siRNA-resistantDDX39B trans-gene, either wild type (FIG. 1E, WT) or helicase mutant(FIG. 1F, D199A), under the control of the tetracycline operator. Toppanels illustrate DDX39B western blot analysis, whereas lower panelsshow RT-PCR analysis of endogenous IL7R transcripts. In all panels thedata is shown as mean±s.d., and statistical significance was assessedusing Student's t-test (*** p≤0.0005; ** p≤0.005; * p≤0.05).

FIG. 2 is a map that shows the variants within or adjacent to the DDX39Blocus strongly associated with MS risk. Each diamond represents avariant analyzed, and the color of the diamonds indicates no association(gray), marginal association (yellow) or strong association (red) withMS risk. The variant rs2523506 is indicated with a larger diamond (seeFIG. 3). Black and blue dotted lines indicate thresholds for marginal(p≤1.0×10⁻²) and strong (p≤5.0×10⁻⁸) association, respectively. Thelocation of the four genes present in this region of chromosome 6 isillustrated at the bottom.

FIGS. 3A to 3C show the DDX39B 5′ UTR variant rs2523506 displaysallele-specific DDX39B protein expression. FIG. 1A is a schematicrepresentation of the DDX39B gene (black), spliced mRNA isoforms (red)and location of MS-associated variants rs2523506, rs2523512 andrs2516478 (asterisks). FIG. 3B shows RT-qPCR quantification of DDX39BmRNA levels in human PBMCs (left), and African (YRI/ESN, middle) andEuropean (IBS, right) LCLs stratified by rs2523506 genotype. Each symbolrepresents cells from one individual, and red lines indicate median andinterquartile range for each group. Samples sizes were: PBMC, CC=32,AC=31, AA=23; YRI/ESN, CC=12, AC=11, AA=2; and IBS, CC=12, AC=12, AA=3.FIG. 3C is a Western blot analysis of DDX39B protein abundance inAfrican (YRI/ESN, left) and European (IBS, right) LCLs stratified byrs2523506 genotype. Panels in FIG. 3C were assembled with differentportions of the same gel. Statistical significance of all measurementswas assessed using Student's t-test (two-sided; *** p≤0.0005; **p≤0.005; * p≤0.05).

FIGS. 4A and 4B show the risk allele of rs2523506 reduces translationalefficiency mediated by DDX39B 5′ UTRs. FIG. 4A is a schematicrepresentation of the different DDX39B 5′ UTR luciferase reporters,which differ by alternative 3′ss in exon 2 and the single nucleotidechange at rs2523506 (C/A). FIG. 4B shows the measurements oftranslational efficiency in transfected HeLa cells. RNA levels andluciferase activity were measured by RT-qPCR and dual Luciferase assays,respectively. Translational efficiency (mean±s.d.) was determined bydividing luciferase activity by RNA levels. Statistical significance wasassessed using Student's t-test (two-sided; ** p≤0.005; * p≤0.05).

FIG. 5 shows the functional interaction between DDX39B and IL7R exon 6variants. The interaction between DDX39B and IL7R was functionallytested in DDX39B-depleted HeLa cells using IL7R splicing reporterscarrying either the risk C allele (IL7R-C; left) or the protective Tallele (IL7R-T; right) of rs6897932. RT-PCR analysis of exon 6 splicing(mean±s.d.) reveals higher exon 6 skipping when the levels of DDX39B arereduced in the context of the risk C allele than of the protective Tallele of rs6897932 (Student's t-test, two-sided; ** p≤0.005).

FIGS. 6A and 6B show that DDX39B controls alternative splicing of IL7Rexon 6 in primary CD4⁺ T cells. Knockdown of DDX39B in primary CD4⁺ Tcells from six donors via lentiviral transduction with two independentshRNA against DDX39B (sh3 and sh5) and a non-targeting control shRNA(NTC). Donors are grouped by IL7R genotype: IL7R-CC (FIG. 6A) andIL7R-CT (FIG. 6B), and each panel illustrates DDX39B western blotanalysis (top) and RT-PCR analysis of IL7R exon 6 splicing (bottom) foreach donor individually. In each panel, the plots on the right show theaverage of % exon 6 skipping for each IL7R genotype (mean±s.d.,Student's t-test: ** p≤0.005; * p≤0.05).

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not limit the invention, except as outlined in the claims.

As used herein, the term “multiple sclerosis” refers to awell-characterized neurological disorder caused by demyelination ofnerve tissue. The term “multiple sclerosis” or “MS” as used hereinincludes a disease identified as having a particular art-known status,e.g., relapsing remitting (RRMS), primary progressive (PPMS) andsecondary progressive (SPMS), wherein the status of MS as definedaccording to these and similar terms would be well understood by one ofordinary skill in the art and according to the description provided inthe Examples included herewith. RRMS, the most common form, ischaracterized by onset of symptoms (relapse) followed by complete ornearly complete remission of the symptoms; with this process repeatingitself with variable length and severity. The length of time of relapsesis variable, as is the remission period. PPMS is characterized by onsetof symptoms without subsequent remission, although the severity andconstellation of symptoms may vary. SPMS is characterized by initialonset similar to RRMS, with progression such that remission of symptomsno longer happens. Symptoms of MS include, but are not limited to,cognitive deficits, motor weakness (often seen as balance andcoordination impairment and ataxia), sensory disturbances (most oftenpain, numbness, and tingling), and visual disturbances (most often opticneuritis and diplopia). Other examples of autoimmune diseases for usewith the present invention include those associated with increasedlevels of soluble IL7R or genetic association with IL7R, e.g., Type Idiabetes, Rheumatoid arthritis, Systemic lupus erythematosus, Atopicdermatitis, Ankylosing spondylitis, Primary biliary cirrhosis, orinflammatory bowel syndromes such as Ulcerative colitis and Crohn'sdisease.

As used herein, the term “linked” refers to a region of a chromosomethat is shared more frequently in family members or members of apopulation affected by a particular disease or disorder, than would beexpected or observed by chance, thereby indicating that the gene orgenes or other identified marker(s) within the linked chromosome regioncontain or are associated with an allele that is correlated with thepresence of a disease or disorder, or with an increased or decreasedrisk of the disease or disorder. Once linkage is established,association studies (linkage disequilibrium) can be used to narrow theregion of interest or to identify the marker (e.g., allele or haplotype)correlated with the disease or disorder.

As used herein, the term “linkage disequilibrium” or “LD” refers to theoccurrence in a population of two linked alleles at a frequency higheror lower than expected on the basis of the allele frequencies of theindividual genes. Thus, linkage disequilibrium describes a situationwhere alleles occur together more often than can be accounted for bychance, which often indicates that the two alleles are physically closeon a DNA strand.

As used herein, the term “genetic marker” refers to a region of anucleotide sequence (e.g., in a chromosome) that is subject tovariability (i.e., the region can be polymorphic for a variety ofalleles). For example, a single nucleotide polymorphism (SNP) in anucleotide sequence is a genetic marker that is polymorphic for two (orin some cases, three or four) alleles. SNPs can be present within acoding sequence of a gene, within noncoding regions of a gene (e.g.,intron) and/or in an intergenic region (e.g., between genes). A SNP in acoding region in which both allelic forms lead to the same polypeptidesequence is termed synonymous and if a different polypeptide sequence isproduced, the alleles of that SNP are non-synonymous. SNPs that are notin protein coding regions can still have effects on transcription factorbinding, RNA splicing, RNA localization, RNA structure, mRNAtranslation, microRNA binding, mRNA stability and/or the sequence of thenon-coding RNA.

Other examples of genetic markers of this invention can include but arenot limited to haplotypes (i.e., combinations of alleles),microsatellites, restriction fragment length polymorphisms (RFLPs),repeats (i.e., duplications), insertions, deletions, etc., as are wellknown in the art.

In the present invention, the term genetic marker is also used todescribe the phenotypic effects of the alleles identified herein asassociated with MS; including IL7R mRNA comprising or lacking exon 6,soluble IL7R (sIL7R) protein and membrane bound IL7R protein, and RNAhelicase DDX39B protein, as described herein.

As used herein, the term “allele” refers to one of two or morealternative forms of a nucleotide sequence at a given position (locus)on a chromosome. Usually alleles are nucleotide sequences in the codingsequence of a gene, but sometimes the term is used to refer to anucleotide sequence in a non-coding sequence. An individual's genotypefor a given gene is the set of alleles it happens to possess.

As used herein, the term “haplotype” refers to a set of singlenucleotide polymorphisms (SNPs) on a single chromatid that arestatistically associated. It is thought that these associations, and theidentification of a few alleles of a haplotype block, can unambiguouslyidentify most other polymorphic sites in its region. Such information isvery valuable for investigating the genetics behind common diseases andis collected by the International HapMap Project. The term “haplotype”is also commonly used to describe the genetic constitution ofindividuals with respect to one member of a pair of allelic genes; setsof single alleles or closely linked genes that tend to be inheritedtogether.

A “subject” in this invention is any animal that is susceptible tomultiple sclerosis as defined herein and can include, for example,humans, as well as animal models of MS such as non-human primates (i.e.macaque, marmoset, etc.), rodents (mouse, rat, etc.) or other animalsthat can be subjected to the experimental autoimmune encephalomyelitis(EAE) or other MS disease models. Subjects of this invention can be maleor female. A subject may be identified as being at risk of developing MSor as having or suspected of having MS by the use of genotypic and/orphenotypic screening.

As used herein, the term “nucleic acids” refers to both RNA and DNA,including cDNA, genomic DNA, mRNA, synthetic (e.g., chemicallysynthesized) DNA and chimeras of RNA and DNA. The nucleic acid can bedouble-stranded or single-stranded. Where single-stranded, the nucleicacid can be a sense strand or an antisense strand. The nucleic acid canbe synthesized using nucleotide analogs or derivatives (e.g., inosine orphosphorothioate nucleotides). Such nucleotides can be used, forexample, to prepare oligonucleotides that have altered base-pairingabilities or increased resistance to nucleases.

As used herein, the term “isolated nucleic acid” refers to a nucleotidesequence (e.g., DNA or RNA) that is not immediately contiguous withnucleotide sequences with which it is immediately contiguous (one on the5′ end and one on the 3′ end) in the naturally occurring genome of theorganism from which it is derived. Thus, in one embodiment, an isolatednucleic acid includes some or all of the 5′ non-coding (e.g., promoter)sequences that are immediately contiguous to a coding sequence. The termtherefore includes, for example, a recombinant DNA that is incorporatedinto a vector, into an autonomously replicating plasmid or virus, orinto the genomic DNA of a prokaryote or eukaryote, or which exists as aseparate molecule (e.g., a cDNA or a genomic DNA fragment produced byPCR or restriction endonuclease treatment), independent of othersequences. It also includes a recombinant DNA that is part of a hybridnucleic acid encoding an additional polypeptide or peptide sequence.Standard recombinant DNA methodologies are used to obtain nucleic acidsencoding nucleic acids, peptides, or proteins, or to incorporate nucleicacids into recombinant expression vectors and introduce the vectors intohost cells. In general, the practice of the present invention employs,unless otherwise indicated, conventional techniques of chemistry,molecular biology, recombinant DNA technology, immunology (especially,e.g., antibody technology), and standard techniques of nucleic acidisolation and/or polypeptide isolation. See, e.g., Sambrook, Fritsch andManiatis, Molecular Cloning: Cold Spring Harbor Laboratory Press (1989);Antibody Engineering Protocols (Methods in Molecular Biology), 510,Paul, S., Humana Pr (1996); Antibody Engineering: A Practical Approach(Practical Approach Series, 169), McCafferty, Ed., Irl Pr (1996);Antibodies: A Laboratory Manual, Harlow et al., C.S.H.L. Press, Pub.(1999); and Current Protocols in Molecular Biology, eds. Ausubel et al.,John Wiley & Sons (1992), relevant portions incorporated herein byreference.

As used herein, the term “isolated” refers to a nucleic acid orpolypeptide that is substantially free of cellular material, viralmaterial, and/or culture medium (when produced by recombinant DNAtechniques), or chemical precursors or other chemicals (when chemicallysynthesized). Moreover, an “isolated fragment” is a fragment of anucleic acid or polypeptide that is not naturally occurring as afragment and would not be found in the natural state. Likewise, anisolated cell refers to a cell that is separated from other cells and/ortissue components with which it is normally associated in its naturalstate.

As used herein, the term “oligonucleotide” refers to a nucleic acidsequence of at least about six nucleotides to about 100 nucleotides, forexample, about 15 to 30 nucleotides, or about 20 to 25 nucleotides,which can be used, for example, as a primer in a PCR amplification or asa probe in a hybridization assay or in a microarray. Oligonucleotidescan be natural or synthetic, e.g., DNA, RNA, modified backbones, etc.Peptide nucleic acids (PNAs) can also be used as probes in the methodsof this invention. The present invention further provides fragments oroligonucleotides of the nucleic acids of this invention, which can beused as primers or probes. Thus, in some embodiments, a fragment oroligonucleotide of this invention is a nucleotide sequence that is atleast, for example 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 1000, 1500, 1750 or 1800contiguous nucleotides of a nucleic acid of this invention (e.g., thegenomic sequence of the IL7R chain gene; the coding sequence or mRNAsequence GenBank Accession No. NM002185 that encodes the IL7R chainprotein (relevant sequence incorporated herein by reference, if morethat one version is available, that available on the date of thisapplication was used), GenBank Accession No. NP002176, with and withoutexon 6 (relevant sequence incorporated herein by reference, if more thatone version is available, that available on the date of this applicationwas used) encoding membrane bound or soluble IL7R chain, respectively,and as taught in U.S. Pat. No. 8,158,344, relevant portions andsequences incorporated herein by reference. Such fragments oroligonucleotides can be detectably labeled or modified, for example, toinclude and/or incorporate a restriction enzyme cleavage site or afluorophore for imaging when employed as a primer in an amplification(e.g., PCR) assay.

The present invention can also include the detection of sIL7R or RNAHelicase DDX39B proteins. Non-limiting examples of protein detectionmethods to detect or measure sIL7R or RNA Helicase DDX39B include, e.g.,LUMINEX, ELISA, immunoassay, mass spectrometry, high performance liquidchromatography, two-dimensional electrophoresis, Western blotting,chemiluminescence immunoassay, a sandwich assay, a precipitationreaction, an immunoprecipitation reaction, a precipitin reaction, a geldiffusion immunodiffusion assay, an agglutination assay, a fluorescentimmunoassay, protein microarray, radioimmunoassay, or antibodymicroarray.

As used herein, the term “biological sample” refers to a sample obtainedfrom a biological subject, including sample of biological tissue orfluid origin including, but limited to, body fluids (e.g., blood, bloodplasma, serum, or urine), secreted bodily fluids such as tears, sputum,rectal or vaginal secretions, or fluids or samples obtained by crossingthe skin such as peritoneal fluids, cerebrospinal fluids, biopsies,organs, tissues, fractions, and cells isolated from the subject.Biological samples may also include extracts from a biological samplethat may comprise proteins and/or nucleic acids.

The present invention can also include the detection of sIL7R or RNAHelicase DDX39B nucleic acids. Non-limiting examples of nucleic acidmethods to detect or measure sIL7R or RNA Helicase DDX39B include, e.g.,a hybridization reaction, a polymerase chain reaction, restrictionendonuclease digestion analysis, Restriction Fragment LengthPolymorphism (RFLP) analysis, an amplification reaction, an isothermalamplification reaction, or a multiplex amplification reaction, apolymerase chain reaction (PCR) amplification reaction, a real-timequantitative polymerase chain reaction (qPCR) amplification reaction, areverse transcriptase PCR (RT-PCR) amplification reaction, primerextension, DNA array technology, a linear amplification technique, aligation reaction, direct sequencing, a sequencing reaction, or acombination thereof.

In the methods described herein, the detection of a genetic marker ofthis invention (e.g., an allele and/or a haplotype) in a subject can becarried out according to methods well known in the art. For example,nucleic acid can be obtained from any suitable sample from the subjectthat will contain nucleic acid and the nucleic acid can then be preparedand analyzed according to well-established protocols for the presence ofgenetic markers according to the methods of this invention. In someembodiments, analysis of the nucleic acid can be carried byamplification of the region of interest according to amplificationprotocols well known in the art (e.g., polymerase chain reaction, ligasechain reaction, strand displacement amplification, transcription-basedamplification, self-sustained sequence replication (3 SR), Qβ replicaseprotocols, nucleic acid sequence-based amplification (NASBA), repairchain reaction (RCR) and boomerang DNA amplification (BDA), etc.). Theamplification product can then be visualized directly in a gel bystaining or the product can be detected by hybridization with adetectable probe. When amplification conditions allow for amplificationof all allelic types of a genetic marker, the types can be distinguishedby a variety of well-known methods, such as hybridization with anallele-specific probe, secondary amplification with allele-specificprimers, by restriction endonuclease digestion, and/or byelectrophoresis. Thus, the present invention further providesoligonucleotides for use as primers and/or probes for detecting and/oridentifying genetic markers, or direct DNA and/or RNA sequencingaccording to the methods of this invention.

The genetic markers of this invention are correlated with multiplesclerosis as described herein according to methods well known in the artand as disclosed in the Examples provided herein for correlating geneticmarkers with various phenotypic traits, including disease states andpathological conditions and levels of risk associated with developing adisease or pathological condition. In general, identifying suchcorrelation involves conducting analyses that establish a statisticallysignificant association and/or a statistically significant correlationbetween the presence of a genetic marker or a combination of markers andthe phenotypic trait in the subject. An analysis that identifies astatistical association (e.g., a significant association) between themarker or combination of markers and the phenotype establishes acorrelation between the presence of the marker or combination of markersin a subject and the particular phenotype being analyzed.

In some embodiments of the methods of this invention, particular allelesof the genetic markers are identified as being correlated with multiplesclerosis or with an increased risk of developing multiple sclerosis.Furthermore, any other allele identified to be highly statisticallycorrelated with this allele can be used to identify a subject atincreased risk of developing MS, such as, a C allele at SNP rs1494555, aC allele at SNP rs987107, an A allele at SNP rs987106, as well as ahaplotype comprising any combination thereof in addition to the C alleleat SNP rs6897932, or any other SNP in LD with rs6897932. Other SNPs ofrelevance to the present invention include rs2523506 (exon 1, 5′ UTR ofDDX39B mRNA), rs2523512 (intron 3) and rs2516478 (intron 9), or anyother SNP in LD with rs2523506.

Furthermore, as described in the Examples section herein, the phenotypicresult of the presence of the C allele at SNP rs6897932 is an increasein the production of an IL7R mRNA lacking exon 6, thereby increasingproduction of the soluble form of the IL7R protein (sIL7R), incombination with the detection of presence of risk alleles in the DDX39Bgene, or a decrease in expression or activity of RNA Helicase DDX39B.

The present inventors further strengthen herein the link between IL7Rexon 6 splicing and MS pathogenesis by demonstrating that trans-actingfactors that control splicing of the exon are candidate genes for MSsusceptibility and treatment. Indeed, the present inventors show hereinthat the RNA helicase DDX39B (previously known as UAP56 and BAT1) is apotent activator of IL7R exon 6, and consequently a repressor of sIL7R,and establish strong genetic association of DDX39B with MS risk.Furthermore, the present inventors show herein that the variantrs2523506 (C/A, where A is the risk allele) in the 5′ UTR of DDX39B mRNAlowers DDX39B protein levels via reduced translation of DDX39B mRNAs,and in doing so it increases MS risk. Critical for the method proposedhere, this DDX39B variant showed strong genetic and functional epistasiswith rs6897932 in IL7R exon 6, wherein carriers of the risk alleles atboth loci are at greater risk to develop the disease, most likely due toincreased skipping of exon 6 and up-regulation of sIL7R.

To date, variants within the Human Leukocyte Antigen (HLA) genes exhibitthe greatest effect on MS risk and are considered the primary geneticdrivers of MS (International Multiple Sclerosis Genetics et al., 2015;Patsopoulos et al., 2013). In contrast, the hundreds of variants outsideof HLA genes identified so far have only small effects on MS risk.Importantly, the joint genotypic effect of this epistatic interactionbetween IL7R rs6897932 and DDX39B rs2523506 is much greater than anyother non-HLA risk variant and comparable to the effect of HLA variants.Therefore, a method is taught herein to identify individuals at highrisk to develop the disease based on genetic screening for allelicvariants in IL7R rs6897932 and DDX39B rs2523506 and phenotypic screeningof IL7R mRNA and protein isoform expression.

Multiple Sclerosis (MS) is an autoimmune disorder where T cells attackneurons in the central nervous system (CNS) leading to demyelination andneurological deficits. A driver of increased MS risk is the soluble formof the interleukin-7 receptor alpha chain gene (sIL7R), produced byalternative splicing of IL7R exon 6. Here, the inventors identified theRNA helicase DDX39B as a potent activator of this exon and consequentlya repressor of sIL7R, and found strong genetic association of DDX39Bwith MS risk. Indeed, the inventors show herein that a genetic variantin the 5′ UTR of DDX39B reduces translation of DDX39B mRNAs andincreases MS risk. Importantly, this DDX39B variant showed stronggenetic and functional epistasis with allelic variants in IL7R exon 6.This study establishes the occurrence of biological epistasis in humansand provides mechanistic insight into the regulation of IL7R exon 6splicing and its impact on MS risk.

MS is characterized by self-reactive T cell mediated damage to neuronalmyelin sheaths in the CNS that leads to axonal demyelination, neuronaldeath and progressive neurological dysfunction. This breach ofimmunological tolerance is thought to originate from complexinteractions between environmental and genetic factors. Addressing thelatter, the inventors the inventors and others uncovered a role for IL7Rin MS susceptibility (Gregory et al., 2007; International MultipleSclerosis Genetics et al., 2007; Lundmark et al., 2007). Together withthe common gamma chain (γ_(c)), IL7R forms a functional cell surfacereceptor for IL7, which is essential for survival, proliferation,maintenance and homeostasis of T cells (Fry and Mackall, 2005;Mazzucchelli and Durum, 2007), and may also be required for optimalTCR-mediated activation of CD4⁺ T cells (Lawson et al., 2015) thought todrive the initial inflammatory phase of MS (Sospedra and Martin, 2005).The skilled artisan will recognize that IL7R refers to the membranebound form of IL7R (also referred to as mIL7R), which is in contrast toa soluble form of IL7R referred to as sIL7R, which is the subject of thepresent invention. Importantly, IL7R expression is precisely anddynamically controlled throughout lymphopoiesis and upon T cellactivation (Alves et al., 2008; Mazzucchelli and Durum, 2007; Munitic etal., 2004; Park et al., 2004), and its modulation has profound effectson immunological function as knockout of IL7R in mice andloss-of-function mutations in humans cause lymphopaenia and severecombined immunodeficiency (Maraskovsky et al., 1996; Peschon et al.,1994; Puel et al., 1998; Roifman et al., 2000). Relevant to theestablishment of self-tolerance, dynamic regulation of IL7R throughoutlymphopoiesis seems critical for selection of self-tolerant T cells(Dooms, 2013).

The single nucleotide polymorphism (SNP) rs6897932 within exon 6 of IL7Ris strongly and reproducibly associated with MS, where the C allele ofthe variant is associated with elevated risk (Gregory et al., 2007;International Multiple Sclerosis Genetics et al., 2007; Lundmark et al.,2007). This variant introduces a non-synonymous threonine to isoleucinechange at amino acid position 244, but this change does not appear toalter IL7 signaling (unpublished results). Importantly, the inventorsshowed that the risk allele enhances skipping of exon 6 (Evsyukova etal., 2013; Gregory et al., 2007), increasing the fraction of mRNAs thatcode for a secreted form of the receptor (sIL7R), and this correlateswith increased plasma levels of sIL7R (Hoe et al., 2010; Lundstrom etal., 2013). Elevated levels of sIL7R have been shown to exacerbate theseverity of experimental autoimmune encephalomyelitis (EAE), a mousemodel of MS, presumably by enhancing the bioactivity or bioavailabilityof IL7 (Lundstrom et al., 2013). These results directly link alternativesplicing of IL7R exon 6 to the pathogenesis of MS.

To better understand how splicing of IL7R exon 6 is regulated, theinventors pursued the discovery of both cis-acting elements andtrans-acting factors controlling splicing of the exon. Previously theinventors identified several important cis-acting elements, among them acritically important exonic splicing enhancer (ESE2), immediatelydownstream of rs6897932, whose mutation (ΔESE2) inhibited inclusion ofexon 6 (Evsyukova et al., 2013). By combining RNA affinitychromatography with mass spectrometry, the inventors discovered factorsinteracting with exon 6 and flanking sequences (Evsyukova et al., 2013)and factors that required ESE2 to bind (Galarza-Munoz et al., 2017). Theinventors showed that knockdown of one of these factors, the DEAD BoxPolypeptide 39B (DDX39B, also known as UAP56/BAT1), increased IL7R exon6 skipping in cell lines and in primary CD4⁺ T cells, and up-regulatedsIL7R secretion. This is relevant in vivo since the inventorsestablished that genetic variants within the DDX39B locus wereassociated with increased genetic risk of MS. The inventors furtherdemonstrated that the risk allele of one of these variants, rs2523506,reduces DDX39B protein level by diminishing the efficiency of DDX39BmRNA translation. Importantly, the increased risk associated with thisvariant showed significant epistasis with rs6897932 in IL7R. This studydemonstrated the occurrence of genetic and functional epistasis of twoMS risk loci in humans and provides a mechanistic explanation for theregulation of IL7R alternative splicing as a driver of MS risk.Furthermore, considering that variants in both IL7R and DDX39B have beenassociated with other autoimmune diseases (Anderson et al., 2011; Cheonget al., 2001; Degli-Esposti et al., 1992; Nakamura et al., 2012;Paternoster et al., 2012; Quinones-Lombrana et al., 2008; Todd et al.,2007), the results of this study could have a broader impact on sharedmechanisms in autoimmunity.

DDX39B is a potent activator of IL7R exon 6. To identify trans-actingfactors controlling splicing of IL7R exon 6, the inventors conducted anunbiased proteomic screen using RNA affinity chromatography and massspectrometry. Using different sets of IL7R exon 6 RNAs and HeLa nuclearextracts the inventors identified a total of 89 candidate trans-factors(Evsyukova et al., 2013). Importantly, the inventors showed that bothHeLa and Jurkat T cells recapitulate the SNP rs6897932- andESE2-dependent changes in IL7R exon 6 splicing, implying exon 6 issimilarly regulated in HeLa and T cells. In the experiments presentedhere, the inventors used RNAs encompassing the first 40 nucleotides (nt)of exon 6, either wild type or ΔESE2 (both containing the C allele ofrs6897932), to identify factors binding in the vicinity of rs6897932 andthe critical ESE2. The inventors identified 66 candidate factors withthese short RNAs, 12 of which showed dependency on ESE2 (Table 1). Thetwo top candidates whose binding was ESE2-dependent were themicrotubule-associated protein 4 (MAP4) and the RNA helicase DDX39B.Functional studies ruled out a role for MAP4 in IL7R exon 6 splicing(data not shown) and thus the inventors focused on DDX39B.

TABLE 1 Trans-acting factors exhibiting dependence on ESE2 for bindingto IL7R exon 6 (Related to FIGS. 1A to 1F). No Protein RNA Wild typeΔESE2 Fold-change Microtubule-associated 1 7 1 −7.0 protein 4 (MAP4) RNAhelicase DDX39B 0 5 1 −5.0 40S Ribosomal protein SA 1 4 1 −4.0 14-3-3protein gamma 0 3 1 −3.0 hnRNP R 0 3 1 −3.0 Scaffold attachment 0 5 2−2.5 factor B1 snRNP E 0 4 2 −2.0 60S Ribosomal Protein L8 0 2 1 −2.0Prothymosin alpha 1 2 1 −2.0 60S Ribosomal Protein L7 0 2 1 −2.0 40SRibosomal protein S3 0 2 1 −2.0 40S Ribosomal Protein S20 0 2 1 −2.0

Trans-acting factors were pulled-down from HeLa nuclear extracts byTobramycin RNA affinity chromatography using an RNA spanning the first40 nt of IL7R exon 6, either wild type or mutant ESE2 (ΔESE2), and a noRNA control (Evsyukova et al., 2013). RNA-Protein complexes were elutedwith excess tobramycin and the bound proteins were identified by massspectrometry. The table indicates the total number of unique peptidescorresponding to each factor that were pulled-down with no RNA control,wild type or ΔESE2 RNAs in two independent experiments, and thefold-change decrease in binding observed with mutation of ESE2.

To determine whether DDX39B regulates splicing of IL7R exon 6, theinventors silenced its expression in HeLa cells using two independentsiRNAs (FIG. 1A), and determined the impact on exon 6 splicing intranscripts from both an IL7R reporter minigene and the endogenous IL7Rgene. Knockdown of DDX39B caused a significant increase in exon 6skipping in transcripts from both the minigene (FIG. 1B) and endogenousgene (FIG. 1C). This effect cannot be explained by differential effectsof DDX39B on stability of IL7R transcript isoforms as both isoformsdecayed with similar rates in control and DDX39B-depleted cells (datanot shown). Importantly, the inventors were able to rescue exon 6splicing in endogenous IL7R transcripts by complementing DDX39B-depletedcells with a siRNA-resistant cDNA trans-gene encoding wild type DDX39B(FIG. 1E) but not a helicase-defective mutant (FIG. 1F). In theseexperiments, DDX39B depletion also led to elevated abundance of overallIL7R transcripts (confirmed by RT-qPCR; data not shown), which may be asecondary effect of DDX39B depletion, as it appeared only at later timepoints during knockdown. Similar rescue experiments conducted with anESE2 mutant reporter strongly suggested there is no DDX39B effect in theabsence of this ESE (FIG. S2). These results indicated the splicingphenotype observed upon transfection of DDX39B siRNAs was specificallydriven by DDX39B protein depletion rather than an off-target effect ofthe siRNAs, and that DDX39B requires its helicase activity and an intactESE2 to activate exon 6 inclusion. Most importantly, the inventorsshowed that DDX39B knockdown elevated the secretion of sIL7R (FIG. 1D).

Previous studies suggested that DDX39B interacts with U2AF65 in thevicinity of the branchpoint sequence (BP) to promote constitutivepre-mRNA splicing (Fleckner et al., 1997; Shen et al., 2008; Shen etal., 2007). Since the data revealed a role for DDX39B in alternativesplicing, the inventors wondered whether DDX39B silencing affects otheralternative splicing events. A pilot investigation in HeLa cellsidentified 75 alternative splicing events that were changed upon DDX39Bdepletion, and a significant fraction were exon skipping events,consistent with DDX39B acting as a splicing activator. The inventorsalso investigated how sensitive inclusion of IL7R exon 6 was tosilencing of other RNA helicases. Knockdown of DDX5, a helicaseimplicated in both transcriptional and post-transcriptional control(Huang et al., 2015), and DDX23, the U5 snRNP-associated human homologueof PRP28 (Teigelkamp et al., 1997), caused small effects in exon 6skipping, whereas knockdown of DDX17, another RNA helicase withalternative splicing activity (Honig et al., 2002) had no effect (FIG.S3). These results suggest that IL7R exon 6 is particularly sensitive toDDX39B levels and indicate that this RNA helicase impacts multiplealternative splicing events. Collectively, these experimentsdemonstrated that DDX39B is an important activator of IL7R exon 6splicing, and a potent repressor of sIL7R.

DDX39B is genetically associated with MS risk. To determine whethergenes encoding regulators of IL7R exon 6 are associated with MSsusceptibility, the inventors performed parallel genetic associationanalyses of autosomal genes encoding candidate trans-acting factorsidentified in the proteomics screen. When candidate factors form part ofmacromolecular complexes, the inventors added the other components ofsuch complexes for a total of 116 candidate genes. The inventorscombined data from six genetic cohorts of non-overlapping subjects ofEuropean descent from previously published meta-analyses (Patsopoulos etal., 2011), which included 4,088 MS cases and 7,444 controls. Genotypedata were available for 4,882 SNPs in 96 of the 116 candidate genes(minor allele frequency (MAF) ≥1%; imputation information score ≥80%).Variants within +/−10 kilobases (kb) of candidate genes were analyzedusing meta-analytic logistic regression models adjusted for populationstratification and cohort origin. A total of 58 SNPs reached genome-widestatistical significance (p≤5.0×10⁻⁸), all of which resided within theDDX39B gene locus.

The DDX39B gene is located within the major histocompatibility complex(MHC). This region harbors the human leukocyte antigen (HLA) genes, theprimary genetic drivers of MS susceptibility (International MultipleSclerosis Genetics et al., 2015; Patsopoulos et al., 2013), and exhibitsextended linkage disequilibrium (LD) (de Bakker et al., 2006).Accordingly, it was imperative to establish whether DDX39B variants wereassociated with increased MS risk independent of the known HLA riskfactors, rather than reporting on HLA risk variants in LD. To this end,the inventors further refined the genetic model to adjust for all knownHLA MS risk variants: HLA-DRB1*15:01, HLA-DRB1*03:01, HLA-DRB1*13:01,HLA-DRB1*04:04, HLA-DRB1*04:01, HLA-DRB1*14:01, HLA-A*02:01, rs9277489,HLA-B*37:01, and HLA-B*38:01 (hereafter referred to as HLA-adjustedmodel). There were 15 variants in DDX39B with strong association with MSrisk after correction for HLA risk alleles (p≤5.0×10⁻⁸) (FIG. 2). Theinventors used this HLA-adjusted model to inform subsequent functionalanalyses of DDX39B variants displaying strong association with MS risk.While there were four genes within the associated region (FIG. 2), thefunctional experiments demonstrating robust repression of sIL7R byDDX39B (FIGS. 1A to 1F) strongly suggested that DDX39B drives thisassociation and reduces MS risk by decreasing sIL7R expression.

Allele-specific DDX39B protein expression. The inventors nextinvestigated whether any of the DDX39B MS-associated variants alteredits activity. Because none of the associated variants are located in thecoding region, the inventors hypothesized that the functional SNP(s) actby regulating DDX39B expression. Three of the associated variants arelocated within the transcriptional unit of the gene: rs2523506 (exon 1),rs2523512 (intron 3) and rs2516478 (intron 9) (FIG. 3A). The inventorstested whether rs2523512 in intron 3 could impact splicing of exons 3and 4, and found no effect (data not shown). The inventors next focusedon rs2523506 located in the 5′ UTR of DDX39B transcripts where it couldalter mRNA levels and/or their translation efficiency. The inventorsfirst asked whether rs2523506 genotype correlated with changes in DDX39Btranscript levels in peripheral blood mononuclear cells (PBMCs) isolatedfrom relapsing-remitting MS patients and healthy controls.Quantification of the DDX39B mRNAs showed that they did not (FIG. 3B,left), indicating that MS risk association could not be explained bydecreased DDX39B mRNA levels.

The inventors next asked whether rs2523506 could influence translationalefficiency of DDX39B mRNAs, which would lead to differential abundancesof DDX39B protein. To address this, the inventors initially mined aproteomics database where relative protein abundances were quantifiedfor 5,953 genes in lymphoblastoid cell lines (LCLs) (Wu et al., 2013).The inventors found reduced DDX39B protein levels in cell linesheterozygous at rs2523506 (AC) compared to cells homozygous for theprotective allele (CC) in LCLs from an African population (Yoruba inIbadan, Nigeria [YRI]). Unfortunately, no data were available for thehomozygous risk allele (AA). This correlation with DDX39B levels was notdetected in LCLs from an European population (Utah residents withancestry from northern and western Europe [CEU]), which also lacked datafor the AA genotype, suggesting that regulation of DDX39B levels iscomplex and may be differentially modified by other loci or non-geneticfactors between these LCL populations. These data demonstrate that therisk allele of rs2523506 can reduce DDX39B protein expression.

To rigorously test the correlation between DDX39B protein levels andrs2523506 alleles the inventors experimentally analyzed 25 LCLs ofAfrican origin (12 CC, 11 AC and 2 AA) and 27 LCLs of European origin(12 CC, 12 AC and 3 AA). The inventors used LCLs from two Africanpopulations, YRI and Esan in Nigeria (ESN), which were entirely distinctfrom the previously analyzed cell lines (Wu et al., 2013), therebyallowing the inventors to test for replication of the correlationbetween rs2523506 and DDX39B protein levels. Given the lack ofcorrelation in CEU LCLs and concerns associated with these cell linesbeing more extensively passaged than the other LCLs (Yuan et al., 2015),the inventors analyzed LCLs established more recently from Iberianpopulations in Spain (IBS). The inventors first checked levels of DDX39BmRNAs and, similar to findings in PBMCs, found no significantdifferences in DDX39B mRNA levels by rs2523506 genotype in either theAfrican (YRI/ESN) or European (IBS) LCLs (FIG. 3B). Additionally, theinventors assessed whether rs2523506 altered expression of genes in thevicinity of DDX39B (MICB, ATP6V1G2, NFKBIL1, LTA, TNF and LTB) in theAfrican LCLs and found no difference in RNA levels by rs2523506genotype. Furthermore, silencing of vicinal genes that were expressed inHeLa cells had no effect on IL7R exon 6 splicing. These analysesindicated rs2523506 does not affect RNA levels of other genes within theassociated region, consistent with this variant driving MS risk via itsimpact on DDX39B protein expression.

The inventors next analyzed DDX39B protein levels by western blot inboth populations of LCL (FIG. 3C). Analysis in the African LCLs revealeda dose-dependent correlation of the risk allele of rs2523506 withreduced DDX39B protein levels, with approximately 25% reduction in AClines and 50% reduction in AA lines compared to CC lines (FIG. 3C,left). The inventors observed a similar correlation in DDX39B proteinlevels in the European LCLs, albeit to a lesser extent, andstatistically significant when comparing CC and AA lines (FIG. 3C,right). Thus, the inventors concluded that the A risk allele ofrs2523506 is associated with decreased DDX39B protein levels. Moreover,given that DDX39B mRNA levels were unaltered by rs2523506 genotype, theinventors hypothesized that the rs2523506 risk allele functions byreducing the DDX39B mRNA translation.

rs2523506 controls translation efficiency of DDX39B mRNAs. Toinvestigate whether rs2523506 influences translational efficiency ofDDX39B transcripts, the inventors generated luciferase reporterscontaining the DDX39B 5′ UTR variants. In addition to the alternativealleles of rs2523506 (C/A), the 5′ UTR of DDX39B transcripts is furthermodified by alternative 3′ splice sites (3′ss) within exon 2 (3′ss-1 and3′ss-2 in FIG. 3A). Therefore, to precisely mimic the naturallyoccurring DDX39B 5′ UTR variants, the inventors generated reportersreflecting the use of the different 3′ss in exon 2 and the alternativealleles of rs2523506 (C/A) (FIG. 4A). These reporters were transientlytransfected into HeLa cells together with a Firefly luciferase (F-Luc)transfection control, and translational efficiency was determined byquantifying mRNA and luciferase levels, each normalized to the F-Luccontrol. This analysis revealed approximately 20-30% reduction intranslational efficiency of the reporters containing the A risk allelecompared to the C allele (FIG. 4B). Importantly, this effect ofrs2523506 on translational efficiency largely explains the reduction inDDX39B protein levels observed between CC and AA LCLs. Taken togetherwith the results in FIGS. 3A to 3C, the inventors concluded that the Arisk allele of rs2523506 decreases DDX39B protein levels by reducing thetranslational efficiency of DDX39B transcripts.

Gene-gene interaction between DDX39B and IL7R. Centered on the findingsthat the risk allele of rs2523506 reduces DDX39B protein level, and boththe risk allele of IL7R rs6897932 and knockdown of DDX39B increasedskipping of IL7R exon 6, the inventors postulated a functionalrelationship between rs6897932 in IL7R and rs2523506 in DDX39B. Theinventors predicted that carriers of risk alleles in both genes (C inIL7R, and A in DDX39B) would exhibit higher risk of MS. The inventorstherefore tested for evidence of multiplicative interaction (additiveincrease on the log odds scale) between rs2523506 and rs6897932 usinglogistic regression modeling. The interaction model included the maineffects for rs2523506 and rs6897932, and was adjusted for populationancestry, cohort origin and all MEW risk variants outside the DDX39Blocus (HLA-adjusted model) (see Table 3). There was a significantinteraction between rs2523506 and rs6897932 (p=0.029), which was furtherinvestigated in rs6897932-stratified analyses with equivalent modeladjustments. This approach revealed strong association between rs2523506and rs6897932 with MS risk (Table 2, top). The inventors observed noelevated MS risk associated with DDX39B rs2523506 among individualshomozygous for the IL7R rs6897932 protective allele (TT, N=742, oddsratio (OR)=0.98, p=0.904), however an effect was observed among IL7Rrs6897932 heterozygotes (CT, N=4251, OR=1.26, p=7.3×10⁻⁴), and themagnitude and significance of the effect was strongest among individualshomozygous for the IL7R rs6897932 risk allele (CC, N=6239, OR=1.39,p=4.4×10⁻⁹). When testing for the reciprocal relationship, that is therisk associated with IL7R rs6897932 in DDX39B rs2523506 stratifiedanalyses (Table 2, middle), the inventors observed a subtle associationof IL7R in non-carriers of the rs2523506 risk allele (CC, N=7834,OR=1.10, p=0.021), and this effect was drastically modified by thepresence of the rs2523506 risk allele (AC, N=3099, OR=1.20, p=5.1×10⁻³;AA, N=299, OR=2.19, p=7.5×10⁻⁴). The inventors further determined thejoint genotypic effect for all combinations of rs2523506 and rs6897932genotypes (Table 2, bottom). This analysis uncovered a strong epistasisbetween these variants as the inventors observed increased MS risk onlyin carriers of at least one copy of the risk alleles at both loci (IL7RCT, DDX39B AC, N=1167, OR=1.32, p=0.023), and this effect was strongestin individuals homozygous for the risk allele at both loci (IL7R CC,DDX39B AA, N=156, OR=2.75, p=4.5×10⁻⁷).

TABLE 2 Robust epistasis between risk alleles of IL7R rs6897932 andDDX39B rs2523506. Association of rs2523506 and MS risk in individualsstratified by rs6897932 genotype IL7R rs6897932 no TT CT CC strat. OR OROR p-value (95% CI) p-value (95% CI) p-value (95% CI) p-value DDX39B0.029 0.98 0.904 1.26 7.3 × 10⁻⁴ 1.39 4.4 × 10⁻⁹ rs2523506 (0.70, 1.37)(1.10, 1.45) (1.25, 1.55) N = 742 N = 4251 N = 6239 Association ofrs6897932 and MS risk in individuals stratified by rs2523506 genotypeDDX39B rs2523506 no CC AC AA strat. OR OR OR p-value (95% CI) p-value(95% CI) p-value (95% CI) p-value IL7R 0.029 1.10 0.021 1.20 5.1 × 10⁻³2.19 7.5 × 10⁻⁴ rs6897932 (1.02, 1.20) (1.06, 1.37) (1.39, 3.46) N =7834 N = 3099 N = 299 Joint genotypic effect of rs6897932 and rs2523506on MS risk IL7R rs6897932 TT CT CC OR OR OR (95% CI) p-value (95% CI)p-value (95% CI) p-value DDX39B CC ref ref 1.02 0.888 1.15 0.193rs2523506 N = 498 (0.82, 1.27) (0.93, 1.43) N = 2963 N = 4373 AC 1.000.986 1.32 0.023 1.53 3.2 × 10⁻⁴ (0.69, 1.45) (1.04, 1.69) (1.21, 1.92)N = 322 N = 1167 N = 1710 AA 0.83 0.738 1.36 0.189 2.75 4.5 × 10 ⁻⁷(0.29, 2.41) (0.86, 2.15) (1.86, 4.08) N = 22  N = 121  N = 156 

Interaction between rs2523506 and rs6897932 and association with MS riskwas investigated using a logistic regression model adjusted forpopulation stratification, cohort origin and HLA risk variants. Theinteraction model was further investigated using analyses stratified byrs6897932 (top) and rs2523506 (middle) genotypes. The bottom panel showsjoint genotypic effect of rs6897932 and rs2523506 on MS risk. Bolded isthe joint effect in homozygous carriers of the risk allele at both loci.

TABLE 3 Multivariable logistic meta-analysis identifies a significantmultiplicative interaction between IL7R rs6897932 and DDX39B rs2523506(Related to Table 2). HLA model w/o interaction term HLA model withinteraction term Variable Beta SE OR p-value Beta SE OR p-value IL7R0.138 0.035 1.15 8.90E−05 0.087 0.042 1.09 0.036 DDX39B 0.269 0.042 1.311.70E−10 0.342 0.054 1.41 1.90E−10 IL7R * DDX39B — — — — 0.144 0.0661.16 0.029 Intercept −0.826 0.063 0.44 1.20E−39 −0.851 0.064 0.431.30E−40 PC1 3.762 0.936 43.03 5.90E−05 3.756 0.936 42.77 6.00E−05 PC29.689 0.958 16132.3 4.60E−24 9.721 0.958 16662.3 3.50E−24 PC3 −1.4180.926 0.24 0.126 −1.403 0.927 0.25 0.13  PC4 2.565 0.915 12.99 5.10E−032.562 0.915 12.96 5.10E−03 PC5 −4.695 0.924 0.01 3.70E−07 −4.704 0.9240.01 3.60E−07 HLA-DRB1*15:01 1.184 0.042 3.27  4.00E−174 1.184 0.0423.27  3.00E−174 HLA-DRB1*03:01 0.281 0.047 1.32 2.40E−09 0.280 0.0471.32 2.70E−09 HLA-DRB1*13:01 −0.098 0.077 0.91 0.202 −0.098 0.077 0.910.201 HLA-DRB1*04:04 0.275 0.100 1.32 5.70E−03 0.274 0.100 1.31 6.00E−03HLA-DRB1*04:01 −0.160 0.068 0.85 0.018 −0.160 0.068 0.85 0.018HLA-DRB1*14:01 −0.509 0.160 0.60 1.50E−03 −0.509 0.160 0.60 1.50E−03HLA-A*02:01 −0.329 0.037 0.72 1.30E−18 −0.331 0.037 0.72 9.50E−19rs9277489 0.275 0.035 1.32 9.60E−15 0.275 0.035 1.32 9.80E−15HLA-B*37:01 0.538 0.114 1.71 2.10E−06 0.535 0.114 1.71 2.50E−06HLA-B*38:01 −0.540 0.154 0.58 4.70E−04 −0.542 0.154 0.58 4.50E−04 CohortANZgene ref — — — ref — — — BWH/MIGEN −0.882 0.056 0.41 1.20E−56 −0.8820.056 0.41 1.00E−56 IMSGC −0.517 0.059 0.60 1.60E−18 −0.517 0.059 0.601.60E−18 GeneMSA US 0.464 0.083 1.59 2.80E−08 0.465 0.084 1.59 2.50E−08GeneMSA 0.266 0.111 1.30 0.016 0.268 0.111 1.31 0.016 NetherlandsGeneMSA 0.568 0.113 1.77 4.80E−07 0.571 0.113 1.77 4.30E−07 SwitzerlandNumber of observations 11232 11232 Log likelihood −6371.5059 −6369.0997LR chi1 (df) 1985.80 (22) 1990.61 (23) Prob > chi2 <0.00005 <0.00005Pseudo R2 0.1348 0.1352

Multivariable logistic regression meta-analyses demonstrate evidence fora significant multiplicative interaction between IL7R rs6897932 andDDX39B rs2523506, adjusted for population substructure, known HLA riskvariants, and cohort of origin. The table illustrates the output foreach adjusted variable with comparison between the two models: the HLAadjusted model without (left) and with (right) the interaction termbetween IL7R rs6897932 and DDX39B rs2523506 (bolded parameters)[Beta=beta coefficient; SE=standard error; OR=odds ratio]. Both modelsare stable and model parameters are consistent, with the exception ofthe significant statistical interaction.

It should be noted that interaction between DDX39B and IL7R was not dueto an interaction between IL7R and HLA-DRB1*15:01, the main geneticdriver of MS (International Multiple Sclerosis Genetics et al., 2015;Patsopoulos et al., 2013), as the inventors observed no evidence for amultiplicative interaction between rs6897932 and HLA-DRB1*15:01 with MSrisk (Table 4). Taken together, the inventors concluded that the riskalleles in DDX39B and IL7R show strong evidence of epistasis, with arobust dose-dependent effect, suggesting that the MS risk associatedwith low levels of DDX39B can be explained by its action on IL7R.

TABLE 4 Lack of evidence for a multiplicative interaction between riskalleles of IL7R rs6897932 and HLA-DRB1*15:01 (Related to Table 2). IL7Rrs6897932 TT CT CC no strat. OR OR OR p-value (95% CI) p-value (95% CI)p-value (95% CI) p-value HLA-DRB1*15:01 0.11 3.75 6.5 × 10⁻¹⁵ 3.50 3.2 ×10⁻⁷¹ 3.11 4.4 × 10⁻⁹² (2.69, 5.22) (3.05, 4.02) (2.78, 3.46)

The inventors tested for evidence of interaction between HLA-DRB1*15:01and rs6897932 using a logistic regression model adjusted for populationstratification, cohort origin and HLA risk variants, including theDDX39B rs2523506 variant (Table 2); results were not significant (nostrat.). Stratification of individuals by rs6897932 genotypes (IL7R-TT(N=742), CT (N=4251) and CC (N=6239)) for analysis of DRB1*15:01revealed strong association with MS across all three strata (note theoverlap in 95% confidence intervals). The results indicate theinteraction between DDX39B and IL7R is not due to an interaction betweenIL7R and HLA-DRB1*15:01, and thus, it is likely that this interactionbetween DDX39B and IL7R could explain part of the heretofore unaccountedheritable risk of MS.

The epistasis between DDX39B rs2523506 and IL7R rs6897932 suggested thepossibility of a functional interplay that could enhance skipping ofIL7R exon 6 in carriers of the risk alleles at both loci. Indeed,knockdown of DDX39B revealed higher skipping of exon 6 in transcriptsfrom a reporter carrying the risk allele of rs6897932 (IL7R-C) comparedto the protective allele (IL7R-7) (FIG. 5). This result is consistentwith exon 6 skipping being augmented by two insults: strengthening of aweak silencer by the risk allele of rs6897932 (Evsyukova et al., 2013;Gregory et al., 2007) and reduced expression of the activator DDX39B.Collectively, the genetic and functional studies indicate that carriersof both IL7R and DDX39B risk alleles are at highest risk of developingMS, very likely due to increased expression of sIL7R.

DDX39B regulates IL7R exon 6 inclusion in primary CD4⁺ T cells. Theinventors then explored the effect of DDX39B knockdown on IL7R exon 6splicing in CD4⁺ T cells isolated from healthy human donors who wereeither CC (FIG. 6A) or CT (FIG. 6B) at rs6897932 in IL7R. The inventorstransduced cells from six donors with lentiviruses expressing twoindependent shRNAs against DDX39B or a non-targeting control shRNA. Eventhough DDX39B knockdown was less efficient in primary CD4⁺ T cells thanin HeLa cells, the inventors observed similar increase in exon 6skipping upon DDX39B depletion (FIGS. 6A and 6B). The inventors alsomeasured sIL7R levels by ELISA and found increased levels of sIL7R uponDDX39B knockdown in cells from all donors with one DDX39B shRNA (sh3)but only in cells from two donors with the second shRNA (sh5). While thelack of correlation of sIL7R levels with exon 6 skipping in cellstransduced with sh5 remains unexplained, the inventors note it could bedue to the fact that the antibody used can detect all IL7R proteinisoforms. Importantly, the inventors observed higher levels of exon 6skipping upon DDX39B depletion in CD4⁺ T cells from donors homozygousfor the risk allele in IL7R (CC) than those from heterozygous (CT)(FIGS. 6A and 6B), corroborating the results obtained with the IL7Rsplicing reporters (FIG. 5). These data are consistent with the observedDDX39B-IL7R epistasis above (Table 2). The inventors concluded DDX39Bplays an important role in promoting IL7R exon 6 inclusion in primaryCD4⁺ T cells, thereby underscoring the importance of DDX39B in MSpathogenesis.

The inventors previously showed that the cis-acting rs6897932 C allelein exon 6 of IL7R, which is strongly associated with MS susceptibility,increases skipping of exon 6 (Evsyukova et al., 2013; Gregory et al.,2007), and this correlates with elevated levels of circulating sIL7R(Hoe et al., 2010; Lundstrom et al., 2013). This is predicted to haveimportant repercussions on the pathogenesis of MS as sIL7R has beenshown to exacerbate the severity of EAE, a mouse model of MS (Lundstromet al., 2013). The inventors have now found that trans-acting factorscontrolling exon 6 splicing are indicative of MS susceptibility. Using amultidisciplinary approach consisting of biochemical, molecular genetic,and human genetic investigations, the inventors show herein that the RNAhelicase DDX39B is: (1) a potent trans-activator of IL7R exon 6 in celllines and primary human CD4⁺ T cells, (2) a repressor of sIL7R, and (3)a novel risk factor for MS. Of the numerous DDX39B variants found to bestrongly associated with MS risk, the inventors identified rs2523506 asa putative functional variant that exhibits strong correlation withreduced DDX39B protein expression in LCLs. Indeed, functional studieswith luciferase reporters showed that the risk allele of this 5′ UTRvariant diminishes the translational efficiency mediated by DDX39B 5′UTRs, thereby directly linking the SNP to the observed changes inprotein levels. Additionally, rs2523506 does not affect expression ofnearby genes, including pro-inflammatory cytokines of the tumor necrosisfactor superfamily (TNF, LTA and LTB). These findings suggest thatincreased MS risk associated with DDX39B could largely result from itsfunction on IL7R splicing.

DDX39B is a DEAD-box protein with known functions in constitutivepre-mRNA splicing (Shen et al., 2008; Shen et al., 2007) and nuclearexport of mRNAs (Luo et al., 2001; Masuda et al., 2005; Strasser et al.,2002). The increased skipping of exon 6 observed upon DDX39B knockdownmay result from other functions of DDX39B, in particular mRNA nuclearexport. This is unlikely, however, as the inventors have previouslyshown that the effects of rs6897932 and ESE2 are recapitulated in invitro splicing assays with HeLa nuclear extracts (Evsyukova et al.,2013), which implies a direct effect on exon 6 splicing rather thannuclear export. Moreover, the role of DDX39B in promoting exon 6inclusion appears to be independent of its function in constitutivepre-mRNA splicing, wherein DDX39B facilitates recruitment of U2 snRNP tothe branchpoint through its interaction with U2AF65. Here, the inventorsshowed that DDX39B requires an intact ESE2 to promote inclusion of exon6, as functionally supported by failure to pull-down DDX39B with AESE2exon 6 RNAs, and the inability of DDX39B to affect exon 6 inclusion inthe absence of ESE2. Based on these results, the inventors propose thatDDX39B binds to ESE2, directly or indirectly, to promote inclusion ofIL7R exon 6, and by doing so it decreases sIL7R expression and reducesMS risk.

Using powerful genetic association analyses that interrogated candidatetrans-acting factors identified in the proteomic screen, the inventorsuncovered DDX39B as the lone candidate trans-factor exhibiting strongassociation with MS risk independently of HLA risk variants.Interestingly, none of the associated variants in the HLA-adjusted modelhad an effect before correction for HLA risk factors, suggesting maskingby HLA associations. A similar phenomenon was previously reported forrs2516489, a variant located in an intergenic region within 10 kb ofDDX39B, which showed association only after adjusting for other HLA riskvariants (Patsopoulos et al., 2013). The authors proposed Simpson'sparadox as a likely explanation, wherein HLA and DDX39B variants areassociated independently but the effect of DDX39B variants is likelymasked by the effects of HLA variants. This masking by HLA variants mayexplain why associations of DDX39B with autoimmune diseases have notbeen as prominent as the inventors would expect. Importantly, theinventors observed dramatic allele-specific variation of DDX39B proteinlevels (up to two-fold) due to rs2523506 genotype among LCLs of Africanorigin, and to a lesser extent among LCLs of European origin, whichmainly results from an effect of rs2523506 on translational efficiency.A perplexing result is the stronger correlation of rs2523506 genotypewith DDX39B protein levels in African than in European LCLs. While acomplete explanation of this finding is not yet at hand, the dataindicate that regulation of DDX39B levels is likely complex, and othergenetic or epigenetic modifiers could titer DDX39B expression oractivity. Nonetheless, it is remarkable that the correlation betweenDDX39B protein levels and rs2523506 genotype was observed by twodistinct experimental methods, namely western blot (this work) and massspectrometry (Wu et al., 2013), and in two populations of LCLs (YRI/ESNand IBS).

An important finding in this study is the epistatic interaction betweenrs2523506 in DDX39B and rs6897932 in IL7R. This interaction was missedin a recent study that investigated potential polygenic interactions inMS between HLA risk alleles and established non-HLA MS risk variants,including IL7R, because non-HLA risk variants within the MHC, such asrs2516489 and rs2523506, were not interrogated (International MultipleSclerosis Genetics et al., 2015). It has been long thought thatfunctional polymorphisms affecting genes within the same gene networkcould enhance disease risk in complex genetic disorders but this hasbeen difficult to prove. For instance, several studies have reportedsuggestive evidence for genetic interactions in several autoimmunediseases including Systemic Lupus Erythematosus (Zhou et al., 2012),Rheumatoid Arthritis (Briggs et al., 2010; Perdigones et al., 2010) andMS (Shahbazi et al., 2011). Nonetheless, all these cases lack functionalvalidation and understanding of the underlying molecular mechanisms.Here, the inventors demonstrated that the epistasis between DDX39Brs2523506 and IL7R rs6897932 confers higher susceptibility to MS in adose-dependent manner. More importantly, the inventors functionallydemonstrated that the risk alleles at both loci work in concert toincrease skipping of IL7R exon 6, thereby enhancing the biogenesis ofsIL7R. This study represents a rare example where the molecularunderpinnings of a pathogenic epistatic interaction are understood andhave been functionally validated.

Lastly, these studies demonstrate a protective role for DDX39B in thepathogenesis of MS, wherein decreased DDX39B expression results inup-regulation of sIL7R via skipping of IL7R exon 6. Although themultidisciplinary study centers on MS, other genetic studies havesuggested associations of variants within DDX39B with other autoimmunediseases including Rheumatoid Arthritis (Kilding et al., 2004; Okamotoet al., 2003; Quinones-Lombrana et al., 2008), Type 1 Diabetes (Barrettet al., 2009; Cheong et al., 2001; Price et al., 2004; Wong et al.,2003) and Atopic Dermatitis (Paternoster et al., 2012); therefore, it islikely that DDX39B play a similar protective role in these diseases.This is supported by findings of elevated plasma levels of sIL7R inpatients of Rheumatoid Arthritis (Badot et al., 2011), Type 1 Diabetes(Monti et al., 2013), and Systemic Lupus Erythematosus (Lauwerys et al.,2014). In addition, DDX39B has also been proposed to negatively regulateproduction of pro-inflammatory cytokines TNFα, and IL6 (Allcock et al.,2001). Collectively, these data imply that DDX39B may play a larger rolethan previously appreciated in the regulation of immune function and inthe development of autoimmunity.

TABLE 5 Oligonucleotide primers used in this study. SEQ ID NO:Primer description Primer sequence  1 DDX39B cloning start codonAAGCTTGCCACCATGGCAGAGAACGATGT GGACAATGAG  2 DDX39B cloning stop codonCTCGAGCTACCGTGTCTGTTCAATGTAGG AGGA  3 T7 forward (splicing)TAATACGACTCACTATAGGGAGA  4 SP6 reverse (splicing) ATTTAGGTGACACTATAGAA 5 IL7R exon 5 forward (splicing) GGCAGCAATGTATGAGATTAAAGTTCG  6IL7R exon 7 reverse (splicing) CAAAGATGTTCCAGAGTCTTCTTATGATC  7IL7R exon 3 forward RT-qPCR CAATATATGTGTGAAGGTTGGAGAA  8IL7R exon 4 reverse RT-qPCR CATTGGCTCCTTCCCGATAG  9GAPDH forward RT-qPCR AGCCACATCGCTCAGACAC 10 GAPDH reverse RT-qPCRGCCCAATACGACCAAATCC 11 DDX39B exon 6 forward RT-qPCRGTGCTACCTTGAGCAAAGAGA 12 DDX39B exon 7 reverse RT-qPCR AACCCATGCAGCGTCAA13 DDX5 exon 13 forward RT-qPCR AGCAAGTGAGCGACCTTATC 14DDX5 exon 14 reverse RT-qPCR CATCCTTCATGCCTCCTCTAC 15DDX17 exon 12 forward RT-qPCR AGGCCAATCAGGCTATCAATC 16DDX17 exon 13 reverse RT-qPCR CTGAAGAAGTGGTCCGGTAAC 17DDX23 exon 16 forward RT-qPCR TCCAAGATGTGTCTATGGTTGTC 18DDX23 exon 17 reverse RT-qPCR GAACACAGCAGAGTCCTCTTT 19MICB exon 4 forward RT-qPCR ATGGAACACAGCGGGAAT 20MICB exon 5 reverse RT-qPCR CATGGCATAGCAGCAGAAAC 21MCCD1 exon 1 forward RT-qPCR CAAAGCAAGCATGGAAGAGC 22MCCD1 exon 2 reverse RT-qPCR CTGCTGCTCCAACAACTCT 23ATP6V1G2 exon 3 forward RT-qPCR AGCTTCTTGGCATGGTCTG 24ATP6V1G2 exon 3 reverse RT-qPCR GATTTCTTTGAGGGAGGGAACT 25NFKBIL1 exon 2 forward RT-qPCR GGGACACGGCACTGCAT 26NFKBIL1 exon 3 reverse RT-qPCR GGAGGGACAGCGGCTTA 27LTA exon 3 forward RT-qPCR CAAACCTGCTGCTCACCT 28LTA exon 4 reverse RT-qPCR GGGACCAGGAGAGAATTGTTG 29TNF exon 3 forward RT-qPCR CAAGCCTGTAGCCCATGTT 30TNF exon 4 reverse RT-qPCR GAGGTACAGGCCCTCTGAT 31LTB exon 3 forward RT-qPCR GGAGCCAGAAACAGATCTCAG 32LTB exon 4 reverse RT-qPCR CTCGTCAGAAACGCCTGTT 33R-Luc ORF forward RT-qPCR CAGTGGTGGGCCAGATGTAAACAA 34R-Luc ORF reverse RT-qPCR TAAGAAGAGGCCGCGTTACCATGT 35F-Luc ORF forward RT-qPCR CGGAAAGACGATGACGGAAA 36F-Luc ORF reverse RT-qPCR CGGTACTTCGTCCACAAACA

TABLE 6 siRNAs used in this study. Number Gene siRNA description SourceCatalog number 1 Non- NSC [All Stars Qiagen SI03650318 silencingNon-silencing control control] 2 DDX39B DDX_3 [Hs_BAT1_11] QiagenSI04368147 3 DDX39B DDX_4 [Hs_BAT1_13] Qiagen SI05107942 4 DDX5 DDX5_1[Hs_DDX5_10] Qiagen SI04178293 5 DDX5 DDX5_2 [Hs_DDX5_11] QiagenSI04228917 6 DDX5 DDX5_3 [Hs_DDX5_13] Qiagen SI04294486 7 DDX17 DDX17_1[Hs_DDX17_4] Qiagen SI00361018 8 DDX17 DDX17_2 [Hs_DDX17_5] QiagenSI03197033 9 DDX17 DDX17_3 [Hs_DDX17_7] Qiagen SI04223198 10 DDX23DDX23_1 [Hs_DDX23_6] Qiagen SI03144743 11 DDX23 DDX23_2 [Hs_DDX23_7]Qiagen SI04190326 12 DDX23 DDX23_3 [Hs_DDX23_8] Qiagen SI04204767 13MICB MICB_1 [Hs_MICB_2] Qiagen SI00082796 14 MICB MICB_2 [Hs_MICB_6]Qiagen SI02636298 15 MICB MICB_3 [Hs_MICB_7] Qiagen SI03083031 16NFKBIL1 NFKBIL1_1 [Hs_NFKBIL1_6] Qiagen SI03239761 17 NFKBIL1 NFKBIL1_2[Hs_NFKBIL1_7] Qiagen SI04231234 18 NFKBIL1 NFKBIL1_3 [Hs_NFKBIL1_8]Qiagen SI04256539 19 LTA LTA_1 = Hs_LTA_1 Qiagen SI00012467 20 LTA LTA_2= Hs_LTA_5 Qiagen SI03024994 21 LTA LTA_3 = Hs_LTA_6 Qiagen SI0307320322 TNF TNF_1 = Hs_TNF_1 Qiagen SI00012439 23 TNF TNF_2 = Hs_TNF_2 QiagenSI00012446

Cell lines and culture conditions. HeLa and Jurkat T cells were obtainedfrom the Duke University Cell Culture Facility. This facility providesauthenticated cell lines free of mycoplasma contamination. HeLa Flp-InT-Rex cell line (Kaiser et al., 2008) was kindly provided by Dr. EDobrikova (Duke University). LCLs were obtained from the CoriellInstitute Cell Repositories. HeLa cells were grown in DMEM medium (LifeTechnologies), whereas Jurkat T cells and LCLs were grown in Advanced1640 RPMI medium (Life Technologies); both medium were supplemented with10% heat-inactivated FBS (Gemini Bio-Products) and 1% antibiotics (PenStrep; Life Technologies).

Primary human CD4⁺ T cells collection and culture. Informed consent wasobtained from healthy volunteers for blood donation. Collection of bloodsamples was in accordance with the research protocol entitled“Characterizing human immune response signatures that predict clinicaloutcomes by isolating PBMCs, serum and plasma from normal donors whichwill be used for optimization, validation and serve as normal controlsfor immune profiling assays” (Protocol ID: Pro00070584, Kent Weinhold,PI) approved by the Institution Review Board at Duke University.Peripheral blood mononucleated cells (PBMCs) were extracted from wholeblood using the Ficoll method and CD4⁺ T cells were further isolatedusing CD4⁺ T cell isolation kit (Miltenyi Biotec). Isolated primary CD4⁺T cells were cultured in Advanced 1640 RPMI medium supplemented with 20%heat-inactivated FBS, 1% antibiotics and 100 ng/mL human recombinantIL-2 (Peprotech). 48 hours prior transduction, CD4⁺ T cells wereactivated with anti-CD3 (50 ng/mL; eBioscience) and anti-CD28 (100ng/mL; BD Biosciences) antibodies in the media.

DNA and RNA collection from human PBMC and LCL. Informed consent wasobtained from RRMS patients and healthy volunteers enrolled in theMURDOCK-MS study. Blood samples were drawn from these individuals forDNA and RNA isolation. Sample collection was in accordance with theresearch protocol associated with this study as approved by theInstitution Review Board at Duke University. Genomic DNA and total RNAwere isolated from whole blood using the QIAamp DNA Blood mini kit(QIAGEN) and PAXgene Blood RNA kit (QIAGEN), respectively. The inventorspoint out that although these RNA samples were isolated from wholeblood, throughout the text the inventors referred to them as isolatedfrom PBMCs, given that the majority of nucleated cells in whole bloodare PBMCs. Additional samples from individuals enrolled at DukeUniversity were isolated from PBMCs using Trizol reagent. For LCLs,total RNA was harvested using the ReliaPrep RNA Cell Miniprep System(Promega). All RNA samples were treated with TURBO DNase (Ambion)according to the manufacturer's protocol.

Generation of stable cell line expressing DDX39B. HeLa cells stablyexpressing an inducible DDX39B cDNA trans-gene were generated using theFlp-In T-Rex system (Life Technologies) as recommended by themanufacturer. HeLa Flp-In T-Rex cell line (Kaiser et al., 2008) waskindly provided to the group by Dr. E Dobrikova (Duke University). Thecoding sequence of DDX39B was amplified with Phusion High-Fidelity DNApolymerase (New England BioLabs) using as template cDNA prepared fromtotal RNA isolated from human PBMCs. Forward and reverse primerscontained HindIII and XhoI restriction sites, respectively. Theresulting PCR amplicon was cloned into pcDNA5/FRT/TO plasmid usingHindIII and XhoI restriction sites and verified by Sanger sequencing.This plasmid was co-transfected with pOG44 plasmid, which encodes theFlp recombinase, into HeLa Flp-In T-Rex cells using Lipofectamine 2000(Life Technologies) according to the manufacturer's instructions.Transfected cells were grown in DMEM medium supplemented with 10% FBSfree of tetracycline (GE Healthcare Life Sciences) underblasticidin/hygromycinB selection for 15 days, and resistant cells wereexpanded and used for subsequent experiments. Expression of thetrans-gene was induced by addition of tetracycline at 50 μg/ml.

Genotyping of human primary cells and LCLs. DNA samples from human PBMCsand primary CD4⁺ T cells were genotyped for DDX39B rs2523506 and IL7Rrs6897932 using TaqMan genotyping assays (Applied Biosystems) or bysequencing. LCLs were previously genotyped in the HapMap project(International HapMap, 2003, 2005; International HapMap et al., 2007).

Transfection of IL7R splicing reporters. IL7R reporter minigenes,previously described in (Gregory et al., 2007), consisted of the genomicregion of IL7R encompassing 614 bp of intron 5, exon 6 and 573 bp ofintron 6, cloned in between constitutive upstream (U) and downstream (D)exons in the pI-11 plasmid. Three versions of the IL7R reporter minigenewere used containing the alternative alleles of rs6897932 (C or T) ormutation of ESE2 in the context of the risk C allele (ΔESE2). These IL7Rsplicing reporters (25 ng per well in 24-well format) were transfectedinto HeLa cells or Jurkat T cells using Lipofectamine 2000 or FuGENE 6(Roche), respectively, according to the manufacturer's recommendations.Total RNA was harvested 48 hours after transfection using Trizol Reagent(Life Technologies). Exon 6 skipping was determined for each reporter byRT-PCR as delineated below.

DDX39B RNAi-mediated knockdown and rescue. siRNA duplexes against DDX39Bwere purchased from Qiagen (DDX_3=Hs_BAT1_11; DDX_4=Hs_BAT1_13) anddiluted to a final concentration of 10 μM upon arrival. Two independentsiRNAs were used to account for potential off-target effects. AllStarsNegative Control siRNA (Qiagen) was used as negative control in allknockdown experiments. Transfections were performed in biologicaltriplicates for each siRNA. 24 hours prior to siRNA transfection, 5×10⁴HeLa cells were seeded in 500 μL of DMEM medium per well in 24-wellplate format. siRNAs were diluted in Opti-MEM I medium (LifeTechnologies) and transfected at final concentration of 50 nM on days 1and 3 post-seeding using Lipofectamine RNAiMax (Life Technologies),following the manufacturer's recommendations. On day 5 post-seeding, thecells were transfected with 25 ng of the corresponding IL7R reporterminigene using Lipofectamine 2000 (Life Technologies) under themanufacturer's recommendations. Two versions of the IL7R reporterminigene were used containing the alternative alleles of rs6897932(IL7R-C and IL7R-T): for the experiments in FIGS. 1A to 1F the inventorsused the IL7R-C reporter, whereas for the experiments in FIG. 5 theinventors used both IL7R-C and IL7R-T reporters. On day 7, cells wereharvested with Trizol Reagent (Life Technologies) or ReliaPrep RNA CellMiniprep System (Promega) for RNA isolation, or with 1×RIPA buffer forprotein extraction. DDX39B depletion was determined by western blottingas delineated below. Percentage IL7R exon 6 skipping was determined byRT-PCR (see section RT-PCR analysis of IL7R splicing). Knockdownexperiments were performed three times with similar results.

For the rescue experiment in FIGS. 1A to 1F, HeLa cells stablyexpressing a DDX39B cDNA trans-gene, either wild type (wt) or a helicasemutant (D199A, previously characterized in (Shen et al., 2008; Shen etal., 2007)), were grown in the absence or the presence of tetracyclineto control expression of the trans-gene. Cells were transfected with thecorresponding siRNAs on days 1, 3 and 5 post-seeding. For thisexperiment the inventors used DDX39B siRNA DDX_4 because it targets the3′ UTR of the endogenous DDX39B transcripts, which is not present intranscripts from the cDNA trans-genes. Cells were harvested on day 9with the ReliaPrep RNA Cell Miniprep System (Promega) for RNA isolationor with 1×RIPA buffer for western blot analysis. These experiments wereperformed at least two times with similar results. For the experimentwith IL7R-ΔESE2 splicing reporter, this reporter was transfected intocells on day 7 and cells were harvested on day 9 as above.

In both knockdown and rescue experiments the relative level of DDX39Bprotein was verified by western blotting. Total protein was harvestedusing 1×RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate,0.1% SDS, and 50 mM Tris-HCl at pH 7.5) freshly supplemented with 1×protease inhibitors (Roche). 10 μg of total protein were loaded per laneon NuPAGE 4%-12% Bis-Tris pre-cast gels (Life Technologies), transferredto nitrocellulose membranes (Whatman), and blotted using standardprotocols with anti-DDX39B rabbit polyclonal antibody (Abcam, ab47955)and anti-hnRNPA1 mouse monoclonal antibody (Abcam, ab5832) as loadingcontrol.

RNAi-mediated knockdown of other genes products. Knockdown of RNAhelicases DDX5, DDX17 and DDX23, and of genes in the vicinity of theDDX39B locus (MICB, NFKBIL1, LTA and TNF) were performed in HeLa cellswith at least two independent siRNAs and the non-silencing control siRNA(NSC). Three additional genes in this region (MCCD1, ATP6V1G2 and LTB)are not expressed in HeLa cells and thus were not tested. siRNAtransfections were carried out as before but at a final siRNAconcentration of 30 nM. Cells were harvested on day 5 post-seeding forRNA isolation using the ReliaPrep RNA Cell Miniprep System. Knockdownlevels were quantified by RT-qPCR and normalized to GAPDH as detailedbelow. IL7R exon 6 skipping was determined by RT-PCR as delineatedbelow. These experiments were performed once in biological triplicates.

Lentiviral packaging. DDX39B shRNA pLKO.1 vector (sh3=TRCN0000286976;sh5=TRCN0000294383) and the Mission non-targeting control shRNA pLKO.1vector (SHC002) were purchased from Sigma-Aldrich. Lentiviral packagingof these shRNA constructs was carried out in 293T cells usingPolyethylenimine (PEI) method. In brief, 7.5×10⁶ cells were seeded in15-cm dishes in DMEM media supplemented with 10% FBS and 1% antibiotics24 hours prior transfection (3 15-cm dishes per construct). Cells wereco-transfected with 17 μg of the corresponding shRNA pLKO.1 vectors orGFP control vector (pLCE), 17 μg of packaging plasmid (pCMVR8.74) and 7μg of VSV-G envelope plasmid (pMD2.G) in serum-free media and 120 μgPEI. DNA-PEI mixes were added drop-wise to the corresponding dishes and18 hours after the medium was replaced with 20 mL fresh DMEM media.After 72 hours, supernatants were collected, filtered through 0.45 μmfilters and concentrated to 6 mL in Amicon Ultra 100K centrifugal filterunits (EMD Millipore). Concentrated lentiviral particles were stored at4° C. overnight.

Transduction of primary human CD4⁺ T cells. Primary CD4⁺ T cells from 6different donors were transduced following modifications to the methodin (Bilal et al., 2015). In brief, 4.0×10⁶ activated primary CD4⁺ Tcells from each donor were transduced with 1 mL of the correspondingconcentrated lentivirus in the presence of 8 μg/mL hexadimethrinebromide (Sigma-Aldrich) at a final density of 1.0×10⁶ cells/mL in RPMImedia supplemented with 20% FBS, 1% antibiotics and 100 ng/mLrecombinant human IL-2. Transductions were carried out for 72 hours,followed by antibiotic selection with puromycin (1.5 μg/mL) for 96hours. Prior to the start of puromycin selection, GFP-transduced cellswere assayed for GFP expression by flow cytometry. Cells were transducedat 50-60% efficiency as determined by % GFP positive cells, and wereenriched to >90% by puromycin selection. 24 hours prior to collection,cells were resuspended in RPMI media without puromycin and IL-2 foranalysis. Supernatants were collected for quantification of sIL7Rsecretion, and cells were harvested for RNA isolation with the ReliaPrepRNA Cell Miniprep System (Promega) or for protein extraction with 1×RIPAbuffer as before. Knockdown of DDX39B was determined by western blottingas before, and by RT-qPCR with normalization to GAPDH. IL7R exon 6skipping and sIL7R secretion were quantified by RT-PCR and ELISA,respectively, as described below.

RT-PCR analyses of IL7R splicing. Total RNA was harvested using eitherTrizol Reagent (Life Technologies) or the ReliaPrep RNA Cell MiniprepSystem (Promega) according to the manufacturer's instructions. IsolatedRNAs were treated with TURBO DNase (Ambion) or DNase I (Promega) todegrade contaminating DNA following the company's recommendations.0.2-1.0 μg of DNase-treated total RNA was used as template for reversetranscription using either the ImProm-II Reverse Transcription System(Promega) or the High Capacity cDNA Reverse Transcription System(Applied Biosystems) and random hexamer primers. PCR reactions wereprepared as follows: 5 μL of the corresponding RT reaction (diluted 1:5)was mixed with 200 nM of forward and reverse primers (see below), 100 nMdNTPs, 50 mM KCl, 2 mM MgCl₂, 0.3 μL of Taq polymerase, and [α-³²P]dCTP(3000 Ci/mmol, 10 mCi/mL, PerkinElmer) at a final concentration of 0.1Ki/μL. PCR primers for minigene constructs (IL7R-C, IL7R-T andIL7R-ΔESE2) were complementary to T7 (forward) and SP6 (reverse)promoter sequences present in the construct, respectively (Gregory etal., 2007). PCR primers for endogenous IL7R were complementary to exon 5(forward) and exon 7 (reverse). PCR products were electrophoresed on 6%non-denaturing polyacrylamide/TBE gels, dried in a gel drier and exposedto a Molecular Dynamics Phosphorimager screen. Quantifications wereperformed using ImageQuant software (Molecular Dynamics). Percentageexon 6 skipping was determined as: [(skipped product)/(included+skippedproducts)×100]. The data are presented as the mean of triplicate samplesand error bars represent standard deviations. Statistical significancein knockdown and rescue studies was assessed using Student's t-test(two-sided).

Quantification of sIL7R by ELISA. Supernatants from the DDX39B knockdownexperiments in HeLa cells and primary CD4⁺ T cells described above werecollected and concentrated to 100 μl by centrifugation in Amicon Ultra3K centrifugal filters (Millipore, UFC500324). Secreted sIL7R wasquantified from the concentrated supernatants by ELISA as in (Crawley etal., 2010). In brief, 96-wells plate (R&D Systems, DY990) were coated at4° C. overnight with a mouse anti-human IL7R monoclonal antibody (R&DSystems, MAB306). The next day, the plates were blocked with 3% BSA inPBS for one hour (room temp), followed by two-hour incubation (roomtemp) with the concentrated supernatants, and four washes with PBS-Tween(0.05%). Detection of bound sIL7R was carried out by one-hour incubation(room temp) with biotinylated goat anti-human IL7R polyclonal antibody(R&D Systems, BAF306), followed by 30-minute incubation (room temp) withstreptavidin-horseradish peroxidase (Millipore, #18-152), and 20-minuteincubation (room temp) with TMB peroxidase substrate (SurModics BioFX,TMBW-1000-01), with four washes with PBS-Tween (0.05%) betweenmanipulations. The reaction was stopped with 1N H₂SO₄, and the productwas visualized in a plate reader at 450 nm. The concentration of sampleswas extrapolated from standard curves of recombinant human IL7R-Fcchimera (R&D Systems, 306-IR-050). Given that knockdown of DDX39Bincreases skipping of IL7R exon 6, the inventors expected secretion ofsIL7R to increase in the knockdown. For this reason, the inventors useda one-sided Student's t-test to assess statistical significance,comparing each independent siRNA against the negative control (FIG. 1D).This experiment was performed twice with similar results.

RT-qPCR expression analyses. Transcript expression analyses in PBMCs andLCLs were carried out by RT-qPCR. The inventors used 0.2-0.7 μg ofDNase-treated RNA as template for reverse transcription using theImProm-II Reverse Transcription System (Promega) and oligo(dT) primers.cDNAs were diluted 1:5 and RT-qPCR reactions were carried out usingPower SYBR Green PCR Master Mix (Applied Biosystems), with 5 μL of thecorresponding cDNA, and 200 nM of the corresponding forward and reverseRT-qPCR primers. The relative abundance of the target genes (DDX39B,MICB, MCCD1, ATP6V1G2, NFKBIL1, LTA, TNF and LTB) was determined withgene-specific assays spanning an exon junction and normalized to GAPDH.The data were stratified by rs2523506 genotypes and normalized tors2523506 CC genotype. Each point in the box plots representsmeasurements in PBMCs or LCLs from one individual and is shown as theaverage of technical triplicates. Statistical significance wasestablished using the Student's t-test (two-sided).

Overall abundance of IL7R transcripts in rescue experiment and knockdownin primary CD4⁺ T cells was determined by RT-qPCR as before with primerscomplementary to the constitutively spliced exons 3 (forward) and 4(reverse). IL7R levels were normalized to GAPDH, and the data is shownas fold-change over the negative control. Statistical significance wasestablished using the Student's t-test (two-sided).

Global splicing analysis by RNA-seq. Global assessment of alternativesplicing events regulated by DDX39B was carried out by DDX39B knockdownfollowed by RNA-seq. Knockdown was performed as before with DDX39BsiRNAs DDX_3 and DDX_4, and control siRNA (NSC) in two independentexperiments. Poly-A⁺ RNA was enriched from 1 μg of total RNA and used astemplate to generate libraries using the Illumina TruSeq platform asrecommended by the manufacturer. Libraries were sequenced on a 2×100format on an Illumina Hi-Seq 1500. Splicing analysis was carried outusing Vast-tools program version 0.2.1 (Irimia et al., 2014) by aligningthe paired-end reads to the vast-tools human database(vastdb.hsa.7.3.14) using the default parameters. Two replicates of eachcondition were compared with the diff function of vast-tools todetermine differential splicing. Two parameters were used to establishdifferential splicing events: E[APsi], which refers to the difference insplicing between the experimental siRNAs (DDX_3 or DDX_4) and controlsiRNA (NSC) (positive and negative values indicate up-regulation anddown-regulation, respectively), and max(x)@P(|ΔPsi|>x)>0.95, whichindicates the change in splicing at 95% confidence level. To control foroff-target effects, the inventors only considered changes in splicingwith max(x)@P(|ΔPsi|>x)>0.95>10.0 with at least one DDX39B siRNA butE[ΔPsi]≥+/−20.0 with both DDX39B siRNAs.

DDX39B western blot analyses in LCLs. Cell lysates were harvested fromeach cell line at two independent times using 1×RIPA buffer as before.Each cell lysate (10 μg of total protein per lane) was blotted twice,and the values presented correspond to the average of the independentmeasurements. To obtain more accurate measurements of relative proteinabundance, DDX39B protein levels were normalized to either hnRNPA1 (FIG.3C) or PTBP1 (not shown), yielding similar results. DDX39B and hnRNPA1were blotted as before, whereas PTBP1 was blotted using custom-madeanti-PTB rabbit serum (Wagner and Garcia-Blanco, 2002). The inventorsused the Student's t-test (two-sided) to assess statistical significanceof the findings.

Luciferase translation efficiency assays. Luciferase reporters weregenerated by cloning the different DDX39B 5′ UTR variants into pcDNA5plasmid containing the coding sequence of Renilla luciferase (R-Luc).DDX39B 5′ UTR variants were synthesized as gBlocks (Integrated DNATechnologies) and cloned immediately upstream of the R-Luc ORF usingHindIII (5′) and KpnI (3′) restriction sites. All constructs wereverified by sequencing. DDX39B 5′ UTR-R-Luc reporters (25 ng/well) wereindependently co-transfected with a Firefly luciferase control (F-Luc,10 ng/well) into HeLa cells in 24-well format using Lipofectamine 2000as indicated by the manufacturer. 24 hours post-transfection celllysates were collected for luciferase assays with Passive Lysis Bufferand Luciferase assays were immediately performed using the DualLuciferase Reporter Assay System (Promega). Total RNA was isolated fromindependent wells and converted into cDNA as above. RNA level forexperimental and control reporters was quantified by RT-qPCR as beforeusing primers complementary to the R-Luc and F-Luc ORFs, respectively.Luciferase activity and RNA levels were determined by normalizing R-Lucsignals to F-Luc signals (R-Luc/F-Luc). Translational efficiency wasthen determined by dividing normalized Luciferase activity by normalizedRNA levels. Statistical significance was assessed by Student's t-test(two-sided).

Genetic association and gene-gene interaction analyses. The inventorsused genotypic data from six genetic cohorts of non-overlapping case andcontrol subjects of European descent, imputed to >37.4 million SNPsacross the genome using BEAGLE and 1000 Genomes Interim Phase Ihaplotypes using standard procedures (Patsopoulos et al., 2011). Theinventors analyzed SNPs within 10 kilobases of autosomal candidate geneswith ≥80% information and minor allele frequency >1% (4882 SNPs in 96 ofthe 116 candidate genes). EIGENSOFT, a principle components algorithm,identified genetic outliers and calculated the top 10 eigenvectors ofthe genotype data within each cohort; the first 5 eigenvectorssufficiently accounted for population stratification (Patsopoulos etal., 2011). Each variant was analyzed using a meta-analytic logisticregression model as implemented in PLINK (Purcell et al., 2007),assuming alleles have an additive effect on the log-odds scale withineach cohort. Each model included the top 5 principal components. Theinventors assumed the genetic effects were fixed across all cohorts, aspreviously performed for these data (Patsopoulos et al., 2013;Patsopoulos et al., 2011). One candidate gene (DDX39B) resides withinthe MHC, therefore meta-analyses for this locus were also adjusted forknown HLA risk variants (HLA-adjusted model, included HLA-DRB1*15:01,HLA-DRB1*03:01, HLA-DRB1*13:01, HLA-DRB1*04:04, HLA-DRB1*04:01,HLA-DRB1*14:01, HLA-A*02:01, rs9277489, HLA-B*37:01, and HLA-B*38:01),and subsequently for an additional non-HLA MHC risk variant, rs2516489(MHC-adjusted model). These risk variants within MHC region were imputedusing BEAGLE (Browning and Browning, 2009; Patsopoulos et al., 2013) byleveraging a collection of 2,767 individuals of the Type 1 DiabetesGenetics Consortium with typed classical HLA alleles as previouslydescribed and validated (International et al., 2010; Patsopoulos et al.,2013; Raychaudhuri et al., 2012). The inventors used a logisticregression model in STATA (StataCorp) to investigate a multiplicativeinteraction between DDX39B rs2523506 and IL7R rs6897932, which wascharacterized further in IL7R rs6897932 and DDX39B rs2523506 stratifiedanalyses. Similar methodology was employed to study interaction betweenHLA-DRB1*15:01 and IL7R rs6897932. The joint genotypic effect ofrs2523506 and rs6897932 on MS risk was determined by comparing eachgenotypic combination to the reference (non-carriers of risk alleles atboth loci). All models were adjusted for population stratification,cohort origin and HLA risk variants.

Molecular analyses. Statistical significance of all molecular analysesin this study was assessed by Student's t-test (two-sided; *** p≤0.0005;** p≤0.005; * p≤0.05). Sample sizes for expression analyses in PBMCs andLCLs are indicated in the text and in the legend of the correspondingfigures. Data on expression analyses in PBMCs and LCLs is shown asmedian with interquartile range, whereas data on knockdown and rescueexperiments is presented as mean±s.d. These measurements are indicatedin the legend of the corresponding figures.

Genetic Association Analyses and gene-gene interaction. Logisticregression models were used to examine the relationship between geneticvariants and multiple sclerosis disease status (binary trait); theinventors report odds ratios and 95% confidence intervals in the text.Genome-wide statistical significance was imposed for these meta-analyticmodels (two-sided; p≤5×10-8). Models for variants residing within theMHC: having met the significance threshold, were subsequently adjustedfor all other known MS MHC risk variants; statistical significance ofp≤0.05 was imposed for these fully parameterized models. Similarsignificance threshold of p≤0.05 was imposed for the interactionsbetween IL7R rs6897932 and DDX39B rs2523506, and HLA-DRB1*15:01 and IL7Rrs6897932. Sample sizes for these analyses are reported in the text.

Data and Software Availability. The accession number for the RNA-seqdataset reported in this paper is GEO: GSE94730.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method, kit, reagent, orcomposition of the invention, and vice versa. Furthermore, compositionsof the invention can be used to achieve methods of the invention.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, numerous equivalents to the specificprocedures described herein. Such equivalents are considered to bewithin the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps. In embodiments of any of the compositions andmethods provided herein, “comprising” may be replaced with “consistingessentially of” or “consisting of”. As used herein, the phrase“consisting essentially of” requires the specified integer(s) or stepsas well as those that do not materially affect the character or functionof the claimed invention. As used herein, the term “consisting” is usedto indicate the presence of the recited integer (e.g., a feature, anelement, a characteristic, a property, a method/process step or alimitation) or group of integers (e.g., feature(s), element(s),characteristic(s), property(ies), method/process steps or limitation(s))only.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

As used herein, words of approximation such as, without limitation,“about”, “substantial” or “substantially” refers to a condition thatwhen so modified is understood to not necessarily be absolute or perfectbut would be considered close enough to those of ordinary skill in theart to warrant designating the condition as being present. The extent towhich the description may vary will depend on how great a change can beinstituted and still have one of ordinary skilled in the art recognizethe modified feature as still having the required characteristics andcapabilities of the unmodified feature. In general, but subject to thepreceding discussion, a numerical value herein that is modified by aword of approximation such as “about” may vary from the stated value byat least ±1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

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What is claimed is:
 1. A method of identifying a human subject as havingan increased risk of developing an autoimmune disease caused by lowerlevels of an RNA Helicase DDX39B, elevated levels of a solubleinterleukin-7 receptor (sIL7R), or both, comprising: obtaining abiological sample from a subject suspected of having an autoimmunedisease; and detecting or measuring in the biological sample an amountof an RNA Helicase DDX39B, the sIL7R, or both, whereby a lowerexpression of DDX39B or elevated expression of sIL7R, or both identifiesthe subject from which the biological sample was obtained as having anautoimmune disease or having increased risk of developing an autoimmunedisease, when compared to a human subject not having an autoimmunedisease.
 2. The method of claim 1, further comprising the step ofdetecting or measuring an expression level of at least one of: thesoluble isoform of an Interleukin-7 Receptor (sIL7R) or the RNA HelicaseDDX39B, wherein a higher expression of sIL7R or a lower expression ofDDX39B identifies the subject from which the biological sample wasobtained as having an autoimmune disease or having increased risk ofdeveloping an autoimmune disease, when compared to a human subject nothaving an autoimmune disease.
 3. The method of claim 1, wherein theautoimmune disease is selected from Multiple sclerosis, Type I diabetes,Rheumatoid arthritis, Systemic lupus erythematosus, Atopic dermatitis,Ankylosing spondylitis, Primary biliary cirrhosis, or inflammatory bowelsyndromes such as Ulcerative colitis and Crohn's disease.
 4. The methodof claim 2, wherein the step of detecting or measuring in the biologicalsample is defined further as being selected from at least one of:detecting a presence of the risk alleles at SNPs associated withincreased risk of multiple sclerosis in DDX39B and IL7R genes, at leastone SNP selected from at least rs6897932 and rs2523506, or any allele inlinkage disequilibrium with the DDX39B and IL7R MS risk alleles; adifferential expression of IL7R RNA isoforms; a differential expressionof IL7R protein isoforms; a differential expression of DDX39B RNA; adifferential expression of DDX39B protein; or any combination thereof;detecting or measuring in the biological sample is defined further asdetecting allelic variants in DDX39B nucleic acids, which encodes an RNAhelicase critical for inclusion of exon 6 in the Interleukin-7 receptor(IL7R) mRNA, whereby the presence of the risk A allele at the SNPrs2523506 in DDX39B exon 1 (5′ UTR of DDX39B mRNAs), or the presence ofthe complementary allele in the opposite strand, or the presence of anyother allele in linkage disequilibrium with rs2523506, identifies thesubject from which the biological sample was obtained as having multiplesclerosis or having an increased risk of developing multiple sclerosis,relative to a biological sample from a human subject lacking the riskallele at the SNP rs2523506; detecting or measuring in the biologicalsample is defined further as detecting allelic variants in DDX39B andthe Interleukin-7 receptor (IL7R), whereby the presence of the risk Aallele at the SNP rs2523506 in DDX39B exon 1 (5′ UTR of DDX39B mRNAs)and the presence of the risk C allele at the SNP rs6897932 in IL7R exon6, or the presence of the complementary allele in the opposite strand,or the presence of any other allele in linkage disequilibrium with atleast one of rs2523506 or rs6897932, identifies the subject from whichthe biological sample was obtained as having multiple sclerosis or anincreased risk of developing multiple sclerosis, relative to abiological sample from a human subject lacking the risk allele at theSNPs rs2523506 and/or rs6897932; detecting or measuring in thebiological sample is defined further as detecting phenotypic differencesin the expression of Interleukin-7 receptor (IL7R) mRNA isoforms and RNAHelicase DDX39B, wherein an elevated fraction of IL7R mRNAs that lackexon 6 or lower levels of expression of the RNA Helicase DDX39B areindicative of having multiple sclerosis or being at increased risk formultiple sclerosis, whereby an elevated fraction of IL7R mRNAs that lackexon 6 or lower levels of expression of the RNA Helicase DDX39B, or bothin the biological sample from an individual carrier of the risk allelesat rs6897932 and/or rs2523506, or any other variant in linkagedisequilibrium with rs6897932 and/or rs2523506, identifies the subjectfrom which the biological sample was obtained as having multiplesclerosis or an increased risk of developing multiple sclerosis,relative to a biological sample from a human subject lacking the riskallele at the SNPs rs2523506 and/or rs6897932; detecting or measuring inthe biological sample is defined further as detecting phenotypicdifferences in the expression of Interleukin-7 receptor (IL7R) proteinisoforms and RNA Helicase DDX39B, wherein elevated levels of the solubleform of IL7R (sIL7R) and/or lower levels of the RNA Helicase DDX39B areindicative of having multiple sclerosis or being at increased risk formultiple sclerosis, whereby elevated levels of sIL7R or lower expressionof the RNA Helicase DDX39B, or both, in the biological sample from anindividual carrier of the risk alleles at either rs6897932 andrs2523506, or any other variant in linkage disequilibrium with rs6897932and/or rs2523506, identifies the subject from which the biologicalsample was obtained as having multiple sclerosis or an increased risk ofdeveloping multiple sclerosis, relative to a biological sample from ahuman subject lacking the risk allele at the SNPs rs2523506 and/orrs6897932; detecting or measuring in the biological sample is definedfurther as detecting phenotypic differences in the expression of DDX39Bprotein in a subject suspected of having multiple sclerosis, wherebydecreased expression of DDX39B protein in the biological sampleidentifies the subject from which the biological sample was obtained ashaving multiple sclerosis or an increased risk of developing multiplesclerosis, relative to a biological sample from a subject not suspectedto have multiple sclerosis; or detecting or measuring in the biologicalsample is defined further as detecting phenotypic differences in theexpression of DDX39B protein, whereby decreased expression of DDX39Bprotein in the biological sample from an individual carrier of the riskallele at rs2523506, or any other variant in linkage disequilibrium withrs2523506, identifies the subject from which the biological sample wasobtained as having multiple sclerosis or an increased risk of developingmultiple sclerosis, relative to a biological sample from a human subjectlacking the risk allele at the SNPs rs2523506.
 5. The method of claim 1,wherein the step of detecting or measuring in the biological sample is adetection of nucleic acids by a hybridization reaction, a polymerasechain reaction, restriction endonuclease digestion analysis, restrictionfragment length polymorphism (RFLP) analysis, an amplification reaction,an isothermal amplification reaction, or a multiplex amplificationreaction, a polymerase chain reaction (PCR) amplification reaction, areal-time quantitative polymerase chain reaction (qPCR) amplificationreaction, a reverse transcriptase PCR (RT-PCR) amplification reaction,primer extension, DNA array technology, a linear amplificationtechnique, a ligation reaction, direct sequencing, a sequencingreaction, or a combination thereof; or the step of detecting ormeasuring in the biological sample is a detection of proteins byLUMINEX, ELISA, immunoassay, mass spectrometry, high performance liquidchromatography, two-dimensional electrophoresis, Western blotting, flowcytometry, chemiluminescence immunoassay, a sandwich assay, aprecipitation reaction, an immunoprecipitation reaction, a precipitinreaction, a gel diffusion immunodiffusion assay, an agglutination assay,a fluorescent immunoassay, protein microarray, radioimmunoassay, orantibody microarray, or both.
 6. The method of claim 2, furthercomprising at least one of: detecting a pre-mRNA, mRNA, or protein ofInterleukin-7 receptor (IL7R) exon 6 splice variants in the biologicalsample; differentiating between a subject having an increased risk ofmultiple sclerosis or as having multiple sclerosis; detecting DDX39Binteraction with ESE2 that promotes inclusion of IL7R exon 6, anddecreases sIL7R expression, which is indicative of a reduced risk formultiple sclerosis; or detecting the presence of the risk allele atrs2523506 in the 5′ UTR of DDX39B, which reduces translation of DDX39BmRNAs and increases MS risk.
 7. The method of claim 1, wherein themethod of identifying a human subject as having an autoimmune disease oran increased risk of developing an autoimmune disease caused by elevatedlevels of soluble Interleukin-7 Receptor (sIL7R) and lower levels of anRNA Helicase DDX39B, comprises: obtaining a biological sample from asubject suspected of having an autoimmune disease; and detecting ormeasuring in the biological sample an amount of a soluble Interleukin-7receptor (sIL7R) and an amount of an RNA Helicase DDX39B, whereby ahigher expression of sIL7R, a lower expression of DDX39B, or both,identifies the subject from which the biological sample was obtained ashaving an autoimmune disease or having increased risk of developing anautoimmune disease, when compared to a human subject not having anautoimmune disease.
 8. An assay comprising: measuring an interactionbetween DDX39B rs2523506 and IL7R rs6897932 by: obtaining a biologicalsample from a subject suspected of having an autoimmune disease;detecting in the biological sample an amount of a soluble Interleukin-7receptor (sIL7R) and an amount of an RNA Helicase DDX39B, whereby higherexpression of sIL7R and/or lower expression of DDX39B identifies thesubject from which the biological sample was obtained as having anincreased risk of developing an autoimmune disease, when compared to ahuman subject not having an autoimmune disease.
 9. The assay of claim 8,wherein the autoimmune disease is selected from Multiple sclerosis, TypeI diabetes, Rheumatoid arthritis, Systemic lupus erythematosus, Atopicdermatitis, Ankylosing spondylitis, Primary biliary cirrhosis, orinflammatory bowel syndromes such as Ulcerative colitis and Crohn'sdisease.
 10. The assay of claim 8, wherein the levels of the sIL7R andRNA Helicase DDX39B are compared to a subject that does not have anautoimmune disease, wherein an increase in sIL7R or a decrease in RNAHelicase DDX39B, or both, are indicative of an increased risk of thesubject having an autoimmune disease.
 11. The assay of claim 8, whereinthe step of detecting or measuring in the biological sample is definedfurther as being selected from at least one of: detecting a presence ofthe risk alleles at SNPs associated with multiple sclerosis in DDX39Band IL7R genes, at least one SNP selected from at least rs6897932 andrs2523506, or any allele in linkage disequilibrium with the DDX39B andIL7R MS risk alleles; a differential expression of IL7R RNA isoforms; adifferential expression of IL7R protein isoforms; a differentialexpression of DDX39B RNA; a differential expression of DDX39B protein;or any combination thereof; detecting or measuring in the biologicalsample is defined further as detecting allelic variants in DDX39Bnucleic acids, which encodes an RNA helicase critical for inclusion ofexon 6 in the Interleukin-7 receptor (IL7R) mRNA, whereby the presenceof the risk A allele at the SNP rs2523506 in DDX39B exon 1 (5′ UTR ofDDX39B mRNAs), or the presence of the complementary allele in theopposite strand, or the presence of any other allele in linkagedisequilibrium with rs2523506, identifies the subject from which thebiological sample was obtained as having multiple sclerosis or having anincreased risk of developing multiple sclerosis, relative to abiological sample from a human subject lacking the risk allele at theSNP rs2523506; detecting or measuring in the biological sample isdefined further as detecting allelic variants in DDX39B and theInterleukin-7 receptor (IL7R), whereby the presence of the risk A alleleat the SNP rs2523506 in DDX39B exon 1 (5′ UTR of DDX39B mRNAs) and thepresence of the risk C allele at the SNP rs6897932 in IL7R exon 6, orthe presence of the complementary allele in the opposite strand, or thepresence of any other allele in linkage disequilibrium with at least oneof rs2523506 or rs6897932, identifies the subject from which thebiological sample was obtained as having multiple sclerosis or anincreased risk of developing multiple sclerosis, relative to abiological sample from a human subject lacking the risk allele at theSNPs rs2523506 and/or rs6897932; detecting or measuring in thebiological sample is defined further as detecting phenotypic differencesin the expression of Interleukin-7 receptor (IL7R) mRNA isoforms,whereby an elevated fraction of IL7R mRNAs that lack exon 6 in thebiological sample from an individual carrier of the risk alleles atrs6897932, rs2523506, or both, or any other variant in linkagedisequilibrium with rs6897932 and/or rs2523506, or the presence of thecomplementary allele in the opposite strand, identifies the subject fromwhich the biological sample was obtained as having multiple sclerosis oran increased risk of developing multiple sclerosis, relative to abiological sample from a human subject lacking the risk allele at theSNPs rs2523506 and/or rs6897932; detecting or measuring in thebiological sample is defined further as detecting phenotypic differencesin the expression of Interleukin-7 receptor (IL7R) protein isoforms,whereby elevated levels of the soluble form of IL7R (sIL7R) in thebiological sample from an individual carrier of the risk allelesrs6897932, rs2523506, or both, or the presence of the complementaryallele in the opposite strand, or any other variant in linkagedisequilibrium with rs6897932, rs2523506, or both identifies the subjectfrom which the biological sample was obtained as having multiplesclerosis or an increased risk of developing multiple sclerosis,relative to a biological sample from a human subject lacking the riskallele at the SNPs rs2523506 and/or rs6897932; detecting or measuring inthe biological sample is defined further as detecting phenotypicdifferences in the expression of DDX39B protein in a subject suspectedof having multiple sclerosis, whereby decreased expression of DDX39Bprotein in the biological sample identifies the subject from which thebiological sample was obtained as having multiple sclerosis or anincreased risk of developing multiple sclerosis, relative to abiological sample from a subject not suspected to have multiplesclerosis; or detecting or measuring in the biological sample is definedfurther as detecting phenotypic differences in the expression of DDX39Bprotein, whereby decreased expression of DDX39B protein in thebiological sample from an individual carrier of the risk allele atrs2523506, or any other variant in linkage disequilibrium withrs2523506, identifies the subject from which the biological sample wasobtained as having multiple sclerosis or an increased risk of developingmultiple sclerosis, relative to a biological sample from a human subjectlacking the risk allele at the SNPs rs2523506; or wherein an allelicvariant of the DDX39B gene is rs2523506 or any other variant in linkagedisequilibrium.
 12. The assay of claim 8, wherein the assay detects ormeasures in the biological sample is a detection of nucleic acids by ahybridization reaction, a polymerase chain reaction, restrictionendonuclease digestion analysis, restriction fragment lengthpolymorphism (RFLP) analysis, an amplification reaction, an isothermalamplification reaction, or a multiplex amplification reaction, apolymerase chain reaction (PCR) amplification reaction, a real-timequantitative polymerase chain reaction (qPCR) amplification reaction, areverse transcriptase PCR (RT-PCR) amplification reaction, primerextension, DNA array technology, a linear amplification technique, aligation reaction, direct sequencing, a sequencing reaction; or theassay detects or measures in the biological sample is a detection ofIL7R or RNA Helicase DDX39B proteins by LUMINEX, ELISA, immunoassay,mass spectrometry, high performance liquid chromatography,two-dimensional electrophoresis, Western blotting, flow cytometry,chemiluminescence immunoassay, a sandwich assay, a precipitin reaction,an immunoprecipitation reaction, a gel diffusion immunodiffusion assay,an agglutination assay, a fluorescent immunoassay, protein microarray,radioimmunoassay, or antibody microarray, or both.
 13. The assay ofclaim 8, comprises a display that shows differentiating between asubject having an increased risk of multiple sclerosis or as havingmultiple sclerosis.
 14. The assay of claim 8, wherein the assay detectsat least one of: DDX39B protein binding to ESE2 that promotes inclusionof IL7R exon 6, and decreases sIL7R expression, which is indicative of areduced risk for multiple sclerosis; or the presence of the risk alleleat rs2523506 in the 5′ UTR of DDX39B, which reduces translation ofDDX39B mRNAs and increases MS risk.
 15. A kit for measuring an RNAHelicase DDX39B, comprising: a container comprising a first agent forthe detection of an amount of an RNA Helicase DDX39B; and instructionsfor determining the amount of the RNA Helicase DDX39B in a biologicalsample.
 16. The kit of claim 15, further comprising instructions fordetermining whether the amount of at least the first agent in abiological sample from a subject that has or is suspected of having anautoimmune disease is greater or lower than an amount in a biologicalsample from a subject that does not have or is not suspected of havingan autoimmune disease.
 17. The kit of claim 15, further comprising atleast one of: reagents for detection of nucleic acids of the RNAHelicase DDX39B by a hybridization reaction, a polymerase chainreaction, restriction endonuclease digestion analysis, restrictionfragment length polymorphism (RFLP) analysis, an amplification reaction,an isothermal amplification reaction, or a multiplex amplificationreaction, a polymerase chain reaction (PCR) amplification reaction, areal-time quantitative polymerase chain reaction (qPCR) amplificationreaction, a reverse transcriptase PCR (RT-PCR) amplification reaction,primer extension, DNA array technology, a linear amplificationtechnique, a ligation reaction, direct sequencing, a sequencingreaction, or a combination thereof; or reagents for detection of the RNAHelicase DDX39B in the biological sample by LUMINEX, ELISA, immunoassay,mass spectrometry, high performance liquid chromatography,two-dimensional electrophoresis, Western blotting, flow cytometry,chemiluminescence immunoassay, a sandwich assay, a precipitationreaction, an immunoprecipitation reaction, precipitin reaction, a geldiffusion immunodiffusion assay, an agglutination assay, a fluorescentimmunoassay, protein microarray, radioimmunoassay, or antibodymicroarray; or reagents for detection of a second agent, wherein thesecond agent is a pre-mRNA, RNA, or protein of the soluble IL7R or themembrane IL7R, and wherein the detection is at the nucleic acid orprotein level.
 18. A method of identifying the activity of an RNAHelicase DDX39B, comprising: obtaining a biological sample; anddetecting or measuring in the biological sample an amount of an RNAHelicase DDX39B, its binding to RNA or DNA, or its activity.
 19. Themethod of claim 18, wherein further comprising measuring an expressionof a soluble Interleukin-7 receptor (sIL7R), wherein the combination ofa higher secretion of sIL7R, a decrease in the expression or activity ofRNA Helicase DDX39B, or both, is determined; or wherein the amount of anRNA Helicase DDX39B is measured by detecting a ratio of IL7R mRNAisoforms including or excluding exon 6, or a ratio of the resulting IL7Rprotein isoforms, or a detectable agent under control of the sequencesthat control splicing of IL7R exon
 6. 20. The method of claim 18,wherein the step of detecting or measuring in the biological sample isat least one of: detection of nucleic acids of the Interleukin-7receptor mRNA lacking exon 6 and the RNA Helicase DDX39B, by ahybridization reaction, a polymerase chain reaction, restrictionendonuclease digestion analysis, restriction fragment lengthpolymorphism (RFLP) analysis, an amplification reaction, an isothermalamplification reaction, or a multiplex amplification reaction, apolymerase chain reaction (PCR) amplification reaction, a real-timequantitative polymerase chain reaction (qPCR) amplification reaction, areverse transcriptase PCR (RT-PCR) amplification reaction, primerextension, DNA array technology, a linear amplification technique, aligation reaction, direct sequencing, a sequencing reaction, or acombination thereof; or detecting or measuring in the biological sampleone or more IL7R protein isoforms and RNA Helicase DDX39B protein byLUMINEX, ELISA, immunoassay, mass spectrometry, high performance liquidchromatography, two-dimensional electrophoresis, Western blotting,chemiluminescence immunoassay, a sandwich assay, a precipitationreaction, an immunoprecipitation reaction, precipitin reaction, a geldiffusion immunodiffusion assay, an agglutination assay, a fluorescentimmunoassay, protein microarray, radioimmunoassay, or antibodymicroarray.